Nanostructured antibacterial and remineralizing dental bonding agents and dental bonding systems

ABSTRACT

The present invention provides dental bonding agents and dental bonding systems comprising the dental bonding agents. The dental bonding agents of the invention are characterized by having antibacterial properties, and in some aspects of the invention, remineralizing properties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant NumberDE017974 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND

Tooth caries are the result of a dietary carbohydrate-modified bacterialinfectious disease, one of the most common bacterial infections inhumans (Loesche, 1986; van Houte, 1994; Featherstone, 2000). The basicmechanism of dental caries is demineralization, or mineral loss, throughattack by acid generated by bacteria (Featherstone, 2004; Deng, 2005;Totiam et al., 2007). Therefore, acidogenic bacteria growth, typicallyin the context of plaque and biofilm formation, is responsible fordental caries (Loesche, 1986; van Houte, 1994; Zero, 1995; Featherstone,2000; Deng et al., 2005; Cenci et al., 2009). Plaque formation has beendescribed to have three steps: pellicle formation, bacteriacolonization, and biofilm maturation (Burne, 1998). In the initialstage, a proteinaceous film called pellicle forms on the tooth surfacewith adsorbed components from saliva, mucosa, and bacteria (Carlen etal., 2001). Bacteria then adhere and colonize on this surface to growinto a biofilm, which is a heterogeneous structure consisting ofclusters of various types of bacteria embedded in an extracellularmatrix (Stoodley et al., 2008). Cariogenic bacteria such asStreptococcus mutans (S. mutans) and lactobacilli in the plaque can takenutrients from carbohydrates and produce organic acids. Acid productioncauses demineralization to the tooth structure beneath the biofilm.

Resin composites have been increasingly used for tooth cavityrestorations because of their aesthetics, direct-filling capability, andenhanced performance (Ferracane, 1995; Bayne et al., 1998; Lim et al.,2002; Ruddell et al., 2002; Watts et al., 2003; Drummond, 2008). Whilethere has been significant improvement in resin compositions, fillertypes, and cure conditions since their introduction (Ruddell et al.,2002; Imazato, 2003; Drummond and Bapna, 2003; Watts et al., 2003; Lu etal., 2005; Xu X et al., 2006; Krämer et al., 2006), formation ofsecondary caries and bulk fracture remain challenges to the use ofresins (Sarrett, 2005; Sakaguchi, 2005). Indeed, resin compositesgenerally do not prevent secondary caries because they do not hinderbacteria colonization and plaque formation. In fact, several studieshave indicated that resin composites have a greater accumulation ofbacteria and plaque than other restorative materials (Svanberg et al.,1990; Imazato et al., 1994; Takahashi et al., 2004). Indeed, caries atthe restoration margins are a frequent reason for replacing existingrestorations (Mjör et al., 2000), accounting for 50-70% of allrestorations (Deligeorgi et al., 2001; Frost, 2002).

Secondary caries may form in the tooth-restoration interface. Dentalbonding systems are used to adhere resin composites to tooth structures(Spencer and Wang, 2002; Park et al., 2009; Pashley et al., 2011), butmicroleakage can allow bacteria to invade the interface. Residualbacteria can also exist in a clean tooth cavity prior to being packedwith the resin composition.

Dental bonding systems that possess antibacterial properties could proveuseful as an additional means of inhibiting the development of secondarycaries by preventing the growth of residual and invading bacteria. Suchantibacterial dental bonding systems would also find use in otherapplications to which dental bonding is employed, including dentinbonding, enamel bonding, tooth roots, marginal repair, as a crowncement, as an inlay/onlay cement, as a pit and fissure sealant, and asan orthodontic bracket adhesive or cement.

SUMMARY

The present invention provides dental bonding agents and dental bondingsystems comprising the dental bonding agents. The dental bonding agentsof the invention are characterized by having antibacterial properties,and in some aspects of the invention, remineralizing properties.

Dental bonding systems employing the dental bonding agents of theinvention can be used in a variety of applications where a bond betweentwo components is required in a dental application. For example, thebonding systems can be prepared for use in dentin bonding, enamelbonding, tooth roots, marginal repair, as a crown cement, as aninlay/onlay cement, as a pit and fissure sealant, and as an orthodonticbracket adhesive or cement. As a specific example, the dental bondingsystems can be used in conjunction with resin composites that are usedto fill voids in teeth after removal of decayed materials. The dentalbonding systems of the invention include a two-component systemcomprising a dental primer and a dental adhesive, a three-step bondingsystem, a two-step bonding system, and a one-step self-adhesive bondingsystem.

The dental bonding agents of the invention include dental primers anddental adhesives. As suggested above, the dental primers and the dentaladhesives are characterized as having antibacterial properties. Theseproperties are imparted by one or more antibacterial agents that areincluded in the dental primers and dental adhesives. In certain aspectsof the invention, the dental primers and dental adhesives may also haveremineralizing properties.

Therefore, in a first embodiment the present invention includes a dentalprimer comprising a primer and one or more antibacterial agents. In oneaspect, the dental primer further comprises a remineralizing agent.

In a second embodiment, the invention includes a dental adhesivecomprising a adhesive and one or more antibacterial agents. In oneaspect, the dental adhesive further comprises a remineralizing agent.

In a third embodiment, the invention includes a dental bonding systemcomprising (i) a dental primer and (ii) a dental adhesive, wherein thedental primer comprises a primer and one or more antibacterial agentsand wherein the dental adhesive comprises an adhesive and one or moreantibacterial agents. In certain aspects, one or both of the dentalprimer and the dental adhesive further comprises a remineralizing agent.In certain aspects, the system further comprises an etchant.

In one aspect, the dental bonding system is a two-component dentalbonding system comprising (i) a dental primer and (ii) a dentaladhesive, wherein the dental primer comprises a primer and one or moreantibacterial agents and wherein the dental adhesive comprises anadhesive and one or more antibacterial agents. In certain aspects, oneor both of the dental primer and the dental adhesive further comprises aremineralizing agent. In certain aspects, the system further comprisesan etchant.

In another aspect, the dental bonding system is three-step dentalbonding system comprising (i) an etchant, (ii) a dental primer, and(iii) a dental adhesive, wherein the dental primer comprises a primerand one or more antibacterial agents and wherein the dental adhesivecomprises an adhesive and one or more antibacterial agents. In certainaspects, one or both of the dental primer and the dental adhesivefurther comprises a remineralizing agent.

In a further aspect, the dental bonding system is two-step dentalbonding system comprising (i) an etchant, and (ii) a mixture comprisinga dental primer and a dental adhesive, wherein the dental primercomprises a primer and one or more antibacterial agents and wherein thedental adhesive comprises an adhesive and one or more antibacterialagents. In certain aspects, one or both of the dental primer and thedental adhesive further comprises a remineralizing agent.

In an additional aspect, the dental bonding system is one-stepself-adhesive bonding system comprising a mixture that comprises anetchant, a dental primer, and a dental adhesive, wherein the dentalprimer comprises a primer and one or more antibacterial agents andwherein the dental adhesive comprises an adhesive and one or moreantibacterial agents. In certain aspects, one or both of the dentalprimer and the dental adhesive further comprises a remineralizing agent.

In each of the embodiments of the present invention, the dental primercomprises (i) one or more primers, and (ii) one or more antibacterialagents. The primers include, but are not limited to, those comprisingBisphenol A diglycidyl methacrylate (Bis-GMA), glycerol dimethacrylate(GDMA), 2-hydroxyethyl methacrylate (HEMA), mono-2-methacryloyloxyethylphthalate (MMEP), methacrylic acid (MA), methyl methacrylate (MMA),4-acryloyloxyethyl trimellitate anhydride (4-AETA), N-phenylglycineglycidyl methacrylate (NPG-GMA), N-tolylglycine glycidyl methacrylate orN-(2-hydroxy-3-((2-methyl-1-oxo-2-propenyl)oxy)propyl)-N-tolyl glycine(NTG-GMA), pyromellitic diethylmethacrylate or2,5-dimethacryloyloxyethyloxycarbonyl-1,4-benzenedicarboxylic acid(PMDM), pyromellitic glycerol dimethacrylate or2,5-bis(1,3-dimethacryloyloxyprop-2-yloxycarbonyl)benzene-1,4-dicarboxylicacid (PMGDM), and triethylene glycol dimethacrylate (TEGDMA). In certainaspects, the primer comprises SCOTCHBOND MULTI-PURPOSE™ (SBMP) primercomprising 35-45% 2-hydroxyethylmethacrylate (HEMA), 10-20% copolymer ofacrylic and itaconic acids, 40-50% water. In certain other aspects, theprimer comprises PMGDM/HEMA at 3.3/1 ratio+1% BAPO+50% acetone.

In each of the embodiments of the present invention, the dental adhesivecomprises (i) one or more adhesives, and (ii) one or more antibacterialagents. The adhesives include, but are not limited to, those comprisingethoxylated bisphenol A glycol dimethacrylate (Bis-EMA), bisphenol Adiglycidyl methacrylate (Bis-GMA), 2-hydroxyethyl methacrylate (HEMA),triethylene glycol dimethacrylate (TEGDMA), urethane dimethacrylate(UDMA), 4-methacryloyloxyethyl trimellitate anhydride (4-META),methacrylic acid (MA), methyl methacrylate (MMA), 4-acryloyloxyethyltrimellitate anhydride (4-AETA), ethyleneglycol dimethacrylate (EGDMA),glycerol dimethacrylate (GDMA), glycerol phosphate dimethacrylate(GPDM), pyromellitic glycerol dimethacrylate or2,5-bis(1,3-dimethacryloyloxyprop-2-yloxycarbonyl)benzene-1,4-dicarboxylicacid (PMGDM). In certain aspects, the adhesive comprises SBMP adhesivecomprising 60-70% BisGMA and 30-40% HEM. In certain other aspects, theadhesive comprises BisGMA/TEGMA at 7/3 ratio+1% BAPO.

The antibacterial agents included in the dental primers and dentaladhesive of the invention include, but are not limited to, antibacterialmonomers, silver-containing nanoparticles (NAg), quaternary ammoniumsalts (QAS), chlorhexidine particles, TiO2 particles and ZnO particles.

The antibacterial monomers include dimethylamino propyl methacrylate(DMAPM), dimethylamino hexyl methacrylate (DMAHM), dimethylamino heptylmethacrylate (DMAHPM), dimethylamino octyl methacrylate (DMAOM),dimethylamino nonyl methacrylate (DMANM), dimethylamino decylmethacrylate (DMADM), dimethylamino undecyl methacrylate (DMAUDM),dimethylamino dodecyl methacrylate (DMADDM), dimethylamino tridecylmethacrylate (DMATDM), dimethylamino tetradecyl methacrylate (DMATTDM),dimethylamino pentadecyl methacrylate (DMAPDM), dimethylamino hexadecylmethacrylate (DMAHDM), dimethylamino heptadecyl methacrylate (DMAHPDM),dimethylamino octadecyl methacrylate (DMAODM), dimethylamino nonadecylmethacrylate (DMANDM), dimethylamino icosyl methacrylate (DMAIOM),dimethylamino henicosyl methacrylate (DMAHOM), and dimethylamino docosylmethacrylate (DMADOM). In certain aspects, the antibacterial monomer isDMADDM, DMATDM, DMATTDM, DMAPDM or DMAHDM.

In certain aspects of the invention, the combined amount of the one ormore antibacterial monomers incorporated into the dental primer ordental adhesive ranges from about 1% to about 50% of the mass of thedental primer or the dental adhesive. In particular aspects, thecombined amount of the one or more antibacterial monomers present in thedental primer or dental adhesive is about 10%, 7.5%, or 5% of the massof the dental primer or dental adhesive.

In certain aspects of the invention, the amount of NAg present in thedental primer or dental adhesive ranges from about 0.05% and about 5% ofthe mass of the dental primer or dental adhesive. In particular aspects,the amount of NAg present in the dental primer or dental adhesive is amass fraction of about 0.1%, 0.25%, or 0.5%.

When present, quaternary ammonium salts may be a mass fraction ofbetween about 1% to 50% of the mass of the dental primer or dentaladhesive, preferably about 3% to about 15% of the mass of the dentalprimer or dental adhesive.

In embodiments where the dental primer or dental adhesive comprises aremineralizing agent, such agents include, but are not limited to,nanoparticles of amorphous calcium phosphate (NACP). In certain aspectsof the invention, the amount of NACP included in the dental primers anddental adhesives ranges from about 10% to about 40% of the mass of thedental primer or dental adhesive. In particular aspects, the massfraction of NACP in the dental primer or dental adhesive is about 20% orabout 30%.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedherein, which form the subject of the claims of the invention. It shouldbe appreciated by those skilled in the art that any conception andspecific embodiment disclosed herein may be readily utilized as a basisfor modifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thatany description, figure, example, etc. is provided for the purpose ofillustration and description only and is by no means intended to definethe limits the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. TEM image of nanoparticles of silver (NAg), and dentin shearbond strength. In (A), the NAg particle sizes ranged from approximately2 to 5 nm, with (mean±sd, n=100) of (2.7±0.6) nm. The NAg particlesappeared to be well dispersed in the resin, without noticeableagglomeration. In (B), the dentin shear bond strength was between 30 to35 MPa. Ten teeth were tested for each group (n=10). The horizontal lineindicates values not significantly different from each other (p>0.1).

FIG. 2. Antibacterial activity of un-cured primers in agar diskdiffusion test. (A-D) Control primer, 10QADM, 0.05NAg, and10QADM+0.05NAg, respectively. Note a small inhibition zone for control,and much wider inhibition zones for primers with QADM and NAg. Thisexample is for mutans streptococci. Total microorganisms and totalstreptococci had similar results. (E-G) Inhibition zone data for totalmicroorganisms, total streptococci, and mutans streptococci,respectively. Each value is mean±sd (n=6). Bars with dissimilar lettersindicate values that are significantly different (p<0.05).

FIG. 3. Biofilm CFU counts and lactic acid production on specimens withcured primers. (A) CFU of total microorganisms, (B) CFU of totalstreptococci, (C) CFU of mutans streptococci, and (D) lactic acidproduction. Each value is mean±sd (n=6). In each plot, values withdissimilar letters are significantly different (p<0.05). Adding QADM orNAg into the primer imparted a potent antibacterial effect. Thestrongest antibacterial effect was achieved with QADM and NAg togetherin the same primer.

FIG. 4. Human dentin shear bond testing: (A) Schematic of specimenpreparation, (B) schematic of shear bond strength testing, (C) shearbond strength data. Ten teeth were used for each group, requiring atotal of sixty third-molars. Each value is mean±sd (n=10). Horizontalline indicates that all six groups had similar shear bond strengths(p>0.1).

FIG. 5. SEM micrographs of dentin-adhesive interfaces: (A) SBMP control,and (B) A&P+10QADM+0.05NAg. Other groups had similar features and arenot included here. “HL” refers to the hybrid layer between the adhesiveand the underlying mineralized dentin. The adhesive resin waswell-infiltrated into dentinal tubules to form resin tags “T”. In (A),the long arrow indicates a long resin tag. The short arrow points to ashort tag, which was shortened due to the sectioning surface not beingparallel to the tubules. Numerous resin tags were observed in allsamples, without noticeable difference between the six groups,indicating that adding QADM and NAg did not affect dentin bonding.

FIG. 6. MTT metabolic activity. Biofilms were grown for 2 d using amicrocosm model. Five adhesive groups were tested following schematic ofFIG. 24B: Control, A+10QADM, A+0.05NAg, A+0.1NAg, A+10QADM+0.05NAg. Onegroup was tested following schematic of FIG. 24A with a primer layer:A&P+10QADM+0.05NAg. Each values is mean±sd (n=6). Values with dissimilarletters are different (p<0.05).

FIG. 7. Microcosm biofilm CFU counts: (A) Total microorganisms, (B)total streptococci, and (C) mutans streptococci. Each values is mean±sd(n=6). Values indicated by dissimilar letters are significantlydifferent (p<0.05). The results showed that: (1) QADM or NAg eachdecreased the CFU compared to commercial adhesive control; (2) higherNAg mass fraction further decreased the CFU; (3) QADM and NAg togetheryielded a greater reduction in CFU than each alone; (4) QADM and NAg inboth primer and adhesive had the strongest antibacterial effect.

FIG. 8. Lactic acid production by dental plaque microcosm biofilms. Eachvalues is mean±sd (n=6). Values indicated by dissimilar letters aresignificantly different (p<0.05).

FIG. 9. Human dentin shear bond strengths. Each value is mean±sd (n=10).The same letter at the bars indicates that all the six groups had bondstrengths that were not significantly different (p>0.1).

FIG. 10. SEM micrographs of dentin-adhesive interfaces. (A) SBMPcontrol, (B) P&A+NAg, A+20NACP, (C) P&A+NAg, A+40NACP. (D) P&A+NAg,A+20NACP at a higher magnification, and (E, F) at even highermagnifications. Adhesives filled the dentinal tubules and formed resintags “T” for all six groups. “HL” indicates the hybrid layer between theadhesive and the underlying mineralized dentin. High magnification SEMin (D-F) revealed numerous NACP nanoparticles in the adhesive layer, inthe hybrid zone, and inside the dentinal tubules. Arrows in (D-F)indicate NACP in the dentinal tubules. NACP were not only able toinfiltrate with the adhesive into straight tubules (E), but also intobent and irregularly-shaped tubules (F).

FIG. 11. MTT assay on metabolic activity of biofilms on specimens of thesix groups (mean±sd; n=6). Control disks had biofilms with a relativelyhigh metabolic activity, indicating no antibacterial effect. However,all the bonding agents modified with NAg and NACP decreased themetabolic activity by more than half. Values with dissimilar letters aresignificantly different (p<0.05).

FIG. 12. Microcosm biofilm CFU per disk for: (A) Total microorganisms,(B) total streptococci, and (C) mutans streptococci (mean±sd; n=6). Ineach plot, values with dissimilar letters are significantly different(p<0.05). The modified bonding systems reduced the CFU counts by aboutan order of magnitude, compared to the commercial control. Therefore,the bonding agents containing NAg and NACP were strongly antibacterial.

FIG. 13. Lactic acid production by biofilms for the six groups (mean±sd;n=6). Acid production by biofilms on disks with NAg plus 30% and 40%NACP were approximately ¼ of that on the control, indicating a potentantibacterial effect of specimens containing NAg and NACP. Values withdissimilar letters are significantly different (p<0.05).

FIG. 14. Dentin Shear Bond Strength of Anti-bacterial, Re-mineralizingPrimer/Adhesive Dental Bonding Agents.

FIG. 15. In (A) and (B), increasing the monomer concentration decreasedthe fibroblast viability (n=6). At each concentration, the antibacterialmonomer with a chain length of 12 (DMADDM) had the highest fibroblastviability (p<0.05). DMADDM and MDPB are less cytotoxic, with higher cellviability, than BisGMA, a monomer commonly used in dental resins. Ineach group of bars in (A), the antibacterials QADM, DMADDM, MDPB andBisGMA appear from left to right, respectively.

FIG. 16. A modified Menschutkin reaction was used to synthesizeantibacterial monomers: (A) DMAHM, and (B) DMADDM.DMAH=N,N-dimethylaminohexane. BEMA=2-bromoethyl methacrylate.DMAHM=dimethylaminohexane methacrylate. DMAD=1-(dimethylamino)docecane.DMADDM=dimethylaminododecyl methacrylate. EtOH=anhydrous ethanol. Thenumber of the alkyl chain length units was 6 for DMAHM and 12 forDMADDM.

FIG. 17. FTIR spectra of reactants and products for: (A) DMAHM, and (B)DMADDM.

FIG. 18. Mechanical properties of composites: (A) flexural strength, and(B) elastic modulus (mean±sd; n=6). NACP+0DMADDM refers to NACPnanocomposite containing 0% DMADDM; NACP+0.75DMADDM refers to NACPnanocomposite containing 0.75% of DMADDM; and so on. Adding up to 3% ofDMADDM into NACP nanocomposite resulted in no significant decrease instrength and elastic modulus. Horizontal line indicates values that arenot significantly different from each other (p>0.1).

FIG. 19. Dental plaque microcosm biofilms adherent on composites: (A)MTT metabolic activity, and (B) lactic acid production (mean±sd; n=6).NACP+0DMADDM refers to NACP nanocomposite containing 0% DMADDM;NACP+0.75DMADDM refers to NACP nanocomposite containing 0.75% of DMADDM;and so on. In each plot, values with dissimilar letters aresignificantly different (p<0.05).

FIG. 20. Colony-forming unit (CFU) counts for: (A) Total microorganisms,(B) total streptococci, and (C) mutans streptococci (mean±sd; n=6).NACP+0DMADDM refers to NACP nanocomposite containing 0% DMADDM;NACP+0.75DMADDM refers to NACP nanocomposite containing 0.75% of DMADDM;and so on. In each plot, values with dissimilar letters aresignificantly different (p<0.05). Note the log scale for the y-axis.

FIG. 21. Measurement of dentin shear bond strength. Extracted humanmolar teeth were used (n=10). Monomer DMADDM and nanoparticles of silver(NAg) were incorporated into a commercial bonding agent SBMP. The bondeddentin samples were immersed in water for 1 day and 6 months, to testthe degradation of bond strength over time. The commercial control lost⅓ of its strength in 6 months.

FIG. 22. In (A) minimum inhibitory concentration (MIC), and in (B)minimum bactericidal concentration (MBC) against S. mutans. A lower MICand MBC indicate a stronger antibacterial potency for the antibacterialagent. CHX had the lowest MIC and MBC. Regarding the two monomers,DMADDM (dimethylaminododecyl methacrylate) with an alkyl chain length of12 was much more potent than DMAHM (dimethylhexylamine methacrylate)with a chain length of 6. The tests were performed in triplicate (n=3).

FIG. 23. Antibacterial activity of un-cured primers against human salivamicrocosm bacteria in agar disk diffusion test (ADT). In the examplesshown in (A), the top two paper disks were impregnated withP+DMADDM+NAg. The middle disk was impregnated with control primer. Thetwo lower disks were impregnated with P+DMADDM. Primer with DMADDM andNAg had strong antibacterial effects against (B) total microorganisms,(C) total streptococci, and (D) mutans streptococci. Each value ismean±sd (n=6). In each plot, values with dissimilar letters aredifferent from each other (p<0.05).

FIG. 24. MTT metabolic activity: (A) Schematic of biofilm on the primercovering the adhesive and composite, (B) biofilm on the adhesive surfacecovering the composite, and (C) MTT metabolic activity. Biofilms weregrown for 2 days using a microcosm model. Groups 1-3, and 6, were testedfollowing schematic of FIG. 24A. Groups 4 and 5 were tested followingschematic of FIG. 24B without a primer layer, to investigate theantibacterial activity of the adhesive. Each values is mean±sd (n=6).Values with dissimilar letters are significantly different from eachother (p<0.05).

FIG. 25. Biofilm CFU on the cured disks of the six groups for: (A) Totalmicroorganisms, (B) total streptococci, and (C) mutans streptococci.Note the log scale for the y-axis. The DMADDM greatly reduced the CFU.Adding DMADDM and NAg together in primer or adhesive was more potentthan using DMADDM alone. Each values is mean±sd (n=6). Values withdissimilar letters are significantly different from each other (p<0.05).

FIG. 26. Lactic acid production by microcosm biofilms adherent on thecured specimens for the six groups. Bars 1-3 are for biofilms on primer,with the adhesive being the unmodified SBMP adhesive. Bars 4 and 5 arefor biofilms on the adhesive without a primer. Bar 6 is for primer andadhesive both being antibacterial. Each value is mean±sd (n=6). Valueswith dissimilar letters are significantly different from each other(p<0.05).

FIG. 27. Dentin shear bond strength. Ten extracted human molars wereused for each group, requiring a total of sixty teeth. Each value ismean±sd (n=10). The horizontal line indicates that the dentin shear bondstrength was not compromised by adding DMADDM and NAg into primer andadhesive (p>0.1).

FIG. 28. Effect of antibacterial monomers from Table 1, with chainlength (CL) of 3, 6, 9, 12, 16 and 18, on early attachment (4 hourculture) and contact-killing of bacteria. Each monomer was mixed intothe primer and adhesive control (P&A control) at 10% mass fraction(mean+−sd; n=6). In the plot, values with dissimilar letters aresignificantly different from each other (p<0.05).

FIG. 29. Effect of antibacterial monomers from Table 1, with chainlength (CL) of 3, 6, 9, 12, 16 and 18, on mature biofilms (48 hours).Each monomer was mixed into the primer and adhesive control (P&Acontrol) at 10% mass fraction (mean+−sd; n=6). Values with dissimilarletters are significantly different from each other (p<0.05). CL=16reduced the biofilm CFU by 4 orders of magnitude, compared to control.

FIG. 30. Effect of mass fraction of the antibacterial monomer DMAHDM(with chain length of 16) on early attachment (4 hour culture) andcontact-killing of bacteria (mean+−sd; n=6). In the plot, values withdissimilar letters are significantly different from each other (p<0.05).

FIG. 31. Effect of mass fraction of the antibacterial monomer DMAHDM(with chain length of 16) on mature biofilms (48 hours) (mean+−sd; n=6).Values with dissimilar letters are significantly different from eachother (p<0.05). DMAHDM at 10% mass fraction reduced the biofilm CFU bynearly 4 orders of magnitude, compared to control.

DETAILED DESCRIPTION

Described herein are novel dental bonding agents, and dental bondingsystems comprising the agents. Dental bonding systems employing thedental bonding agents of the invention can be used in a variety ofapplications where a bond between two components is required in a dentalapplication. For example, the bonding systems can be prepared for use indentin bonding, enamel bonding, tooth roots, marginal repair, as a crowncement, as an inlay/onlay cement, as a pit and fissure sealant, and asan orthodontic bracket adhesive or cement.

The dental bonding agents of the invention include dental primers anddental adhesives. Both the dental primers and the dental adhesives areunique in that they have antibacterial properties. These properties areimparted by one or more antibacterial agents that may be included in thedental primers and the dental adhesives. In certain aspects of theinvention, the dental primers and dental adhesives may also haveremineralizing properties.

The dental primers of the present invention comprise any primer, orcombination of primers, that is suitable for dental use in a subject,such as a human, and one or more antibacterial agents. Suitable primerswill be those commonly used in dental applications. Exemplary primerscomprise Bisphenol A diglycidyl methacrylate (Bis-GMA), glyceroldimethacrylate (GDMA), 2-hydroxyethyl methacrylate (HEMA),mono-2-methacryloyloxyethyl phthalate (MMEP), methacrylic acid (MA),methyl methacrylate (MMA), 4-acryloyloxyethyl trimellitate anhydride(4-AETA), N-phenylglycine glycidyl methacrylate (NPG-GMA),N-tolylglycine glycidyl methacrylate orN-(2-hydroxy-3-((2-methyl-1-oxo-2-propenyl)oxy)propyl)-N-tolyl glycine(NTG-GMA), pyromellitic diethylmethacrylate or2,5-dimethacryloyloxyethyloxycarbonyl-1,4-benzenedicarboxylic acid(PMDM), pyromellitic glycerol dimethacrylate or2,5-bis(1,3-dimethacryloyloxyprop-2-yloxycarbonyl)benzene-1,4-dicarboxylicacid (PMGDM), and triethylene glycol dimethacrylate (TEGDMA). In oneexample, a suitable primer comprises SBMP primer comprising 35-45%2-hydroxyethylmethacrylate (HEMA), 10-20% copolymer of acrylic anditaconic acids, 40-50% water. In another example, the primer comprisesPMGDM/HEMA at 3.3/1 ratio+1% BAPO+50% acetone.

Similarly, the dental adhesives of the present invention comprise anyadhesive, or combination of adhesives, that is suitable for dental usein a subject, such as a human, and one or more antibacterial agents.Suitable adhesives will be those commonly used in dental applications.Exemplary adhesives comprise ethoxylated bisphenol A glycoldimethacrylate (Bis-EMA), bisphenol A diglycidyl methacrylate (Bis-GMA),2-hydroxyethyl methacrylate (HEMA), triethylene glycol dimethacrylate(TEGDMA), urethane dimethacrylate (UDMA), 4-methacryloyloxyethyltrimellitate anhydride (4-META), methacrylic acid (MA), methylmethacrylate (MMA), 4-acryloyloxyethyl trimellitate anhydride (4-AETA),ethyleneglycol dimethacrylate (EGDMA), glycerol dimethacrylate (GDMA),glycerol phosphate dimethacrylate (GPDM), pyromellitic glyceroldimethacrylate or2,5-bis(1,3-dimethacryloyloxyprop-2-yloxycarbonyl)benzene-1,4-dicarboxylicacid (PMGDM). In one example, a suitable adhesive comprises SBMPadhesive comprising 60-70% BisGMA and 30-40% HEM. In another example,the adhesive comprises BisGMA/TEGMA at 7/3 ratio+1% BAPO.

The antibacterial agents included in the dental primers and dentaladhesives of the invention include, but are not limited to,antibacterial monomers, silver-containing nanoparticles (NAg),quaternary ammonium salts (QAS), chlorhexidine particles, TiO2 particlesand ZnO particles.

The antibacterial monomers differ based on the length of the alkyl chainand include dimethylamino propyl methacrylate (DMAPM), dimethylaminohexyl methacrylate (DMAHM), dimethylamino heptyl methacrylate (DMAHPM),dimethylamino octyl methacrylate (DMAOM), dimethylamino nonylmethacrylate (DMANM), dimethylamino decyl methacrylate (DMADM),dimethylamino undecyl methacrylate (DMAUDM), dimethylamino dodecylmethacrylate (DMADDM), dimethylamino tridecyl methacrylate (DMATDM),dimethylamino tetradecyl methacrylate (DMATTDM), dimethylaminopentadecyl methacrylate (DMAPDM), dimethylamino hexadecyl methacrylate(DMAHDM), dimethylamino heptadecyl methacrylate (DMAHPDM), dimethylaminooctadecyl methacrylate (DMAODM), dimethylamino nonadecyl methacrylate(DMANDM), dimethylamino icosyl methacrylate (DMAIOM), dimethylaminohenicosyl methacrylate (DMAHOM), and dimethylamino docosyl methacrylate(DMADOM). The alkyl chain lengths (CL) of the monomers are provided inTable 2 of the Examples. These antibacterial monomers are well suitedfor use in dental primers and dental adhesives that are used in dentalapplications. In certain aspects, the antibacterial monomer is DMADDM(alkyl chain length (CL)=12), DMATDM (CL=13), DMATTDM (CL=14), DMAPDM(CL=15), or DMAHDM (CL=16).

The antibacterial monomers can be incorporated into the dental primer ordental adhesive at antibacterial monomer mass fractions ranging fromabout 0.5% to about 50% of the dental primer or dental adhesive,preferably from about 2% to about 20% of the mass of the dental primeror dental adhesive. In certain aspects, the antibacterial monomers canbe incorporated into the dental primer or dental adhesive atantibacterial monomer mass fractions ranging from about 1% to about 50%,from about 1% to about 25%, from about 2% to about 20%, from about 2.5%to about 25%, from about 2.5% to about 20%, from about 2.5% to about15%, from about 2.5% to about 12.5%, from about 5% to about 25%, fromabout 5% to about 20%, from about 5% to about 15%, from about 5% toabout 10%, from about 7.5% to about 25%, from about 7.5% to about 20%,about 7.5% to about 15% or from about 7.5% to about 12.5% of the mass ofthe dental primer or dental adhesive. In particular aspects, the amountof the antibacterial monomers present in the dental primer or dentaladhesive is a combined amount of about 0.5, 1, 2, 2.5, 3, 4, 5, 6, 7,7.5, 8, 9, 10, 11, 12, 12.5, 13, 14, 15, 16, 17, 17.5, 18, 19, 20, 21,22, 22.5, 23, 24, 25, 26, 27, 27.5, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50% of the mass ofthe dental primer or dental adhesive. In certain aspects of theinvention, the combined amount of the antibacterial monomersincorporated into the dental primer or dental adhesive ranges from about1% to about 50% of the mass of the dental primer or dental adhesive. Inparticular aspects, the combined amount of the antibacterial monomerspresent in the dental primer or dental adhesive is about 10%, 7.5%, or5% of the mass of the primer or adhesive. In certain aspects, one of theantibacterial monomers identified herein is included in a dental primeror dental adhesive. In certain other aspects, two, three, four, or moreof the antibacterial monomers identified herein are included in a dentalprimer or dental adhesive.

Suitable silver-containing nanoparticles (NAg) include, but are notlimited to, silver 2-ethylhexanoate salt, silver-containing glassparticles and silver benzoate. In addition to silver salts, pre-formedsilver nanoparticles can be used. When present, NAg may make up betweenabout 0.001% and about 20% of a mass fraction of the dental primer ordental adhesive. In certain aspects, NAg will make up between about0.001% and about 3%, about 0.05% and about 1%, about 0.05% and about 2%,about 0.05% and about 5%, about 0.08% and about 10%, or about 0.1% andabout 0.5%, of a mass fraction of the dental primer or dental adhesive,or about 0.01%, 0.08%, 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45%,0.50%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5% or 5.0% of a massfraction of the dental primer or dental adhesive. In one aspect, NAgmakes up about 0.1% of a mass fraction of the dental primer or dentaladhesive. The silver particle size can range from about 1 nm to about1000 nm, and in one aspect, from about 2 nm to about 500 nm. In certainaspects, the amount of NAg present in the dental primer or dentaladhesive ranges from about 0.05% and about 5% of the mass of the dentalprimer or dental adhesive. In particular aspects, the amount of NAgpresent in the dental primer or dental adhesive is a mass fraction ofabout 0.1%, 0.25%, or 0.5%.

Suitable quaternary ammonium salts (QASs) include both polymerizablemonomers and non-polymerizable small molecules, and include, but are notlimited to, bis(2-methacryloyloxy-ethyl) dimethyl-ammonium bromide(QADM), methacryloyloxydodecylpyridinium bromide, methacryloxylethylbenzyl dimethyl ammonium chloride, methacryloxylethyl m-chloro benzyldimethyl ammonium chloride, methacryloxylethyl cetyl dimethyl ammoniumchloride, cetylpyridinium chloride, and methacryloxylethyl cetylammonium chloride, QAS chlorides, QAS bromides, QAS monomethacrylates,QAS dimethacrylates, and pre-fabricated QAS particles. When present, theQAS may make up between about 1% and about 50% of a mass fraction of thedental primer or dental adhesive. In certain aspects, the QAS will makeup between about 2% and about 25%, about 3% and about 15%, about 5% andabout 20%, or about 7.5% and about 15% of a mass fraction of the dentalprimer or dental adhesive, or about 1%, 2.5%, 5%, 7.5%, 10%, 12.5, 15%,17.5%, 20%, 22.5%, 25%, 27.5% or 30% of a mass fraction of the dentalprimer or dental adhesive.

In embodiments where the dental primer or dental adhesive comprises aremineralizing agent, such agents include, but are not limited to,nanoparticles of amorphous calcium phosphate (NACP). NACP comprisesnanometer-sized amorphous calcium phosphate (Ca₃[PO₄]₂) particles thatcan be used to produce a dental primer or dental adhesive with high Caand PO₄ release, improved mechanical properties, and improvedantibacterial properties. The dental primers and dental adhesives thatinclude NACP exhibit greatly increased ion release at acidic, cariogenicpH, when these ions are most needed to inhibit caries.

The NACP included in the dental primers and dental adhesives of thepresent invention, when present, will vary in size, but at least about50, 55, 60, 65, 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98 or 99% of the particles, or all of the particles (100%), havean average diameter of between about 10 nm and about 500 nm. In certainaspects, the average diameter will be between about 25 nm and about 400nm, about 50 nm and about 300 nm, about 75 nm and about 200 nm, or about100 nm and about 150 nm. In a particular aspect, the NACP particles havean average diameter of between about 50 nm and about 200 nm.

The amount of NACP included in the dental primers and dental adhesivesmay vary, but the NACP will generally comprise about 1% to about 90% ofthe mass of the dental primer or dental adhesive. In certain aspects,the NACP will comprise about 1% to about 40%, about 5% to about 70%,about 5% to about 60%, about 5% to about 50%, about 5% to about 45%,about 5% to about 40%, about 5% to about 30%, about 10% to about 90%,about 10% to about 80%, about 10% to about 70%, about 10% to about 60%,about 10% to about 50%, about 10% to about 40%, about 15% to about 70%,about 15% to about 60%, about 15% to about 50%, about 15% to about 40%,about 20% to about 90%, about 85% to about 70%, about 20% to about 80%,about 20% to about 70%, about 20% to about 60%, about 20% to about 50%,about 20% to about 40%, about 25% to about 70%, about 25% to about 60%,about 25% to about 50%, about 25% to about 40%, about 30% to about 90%,about 30% to about 85%, about 30% to about 80%, about 30% to about 75%,about 30% to about 70%, about 30% to about 60%, about 15% to about 45%,about 15% to about 35%, or about 15% to about 25% of the mass of thedental primer or dental adhesive. In certain other aspects, the NACP isa mass fraction of about 25%, about 25.5%, about 26%, about 26.5%, about27%, about 27.5%, about 28%, about 28.5%, about 29%, about 29.5% orabout 30% of the dental primer or dental adhesive. In particularaspects, the NACP will comprise about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5,25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89 or 90% of the mass of the dental primer or dental adhesive. Incertain aspects, the NACP will range from about 10% to about 40% of themass of the dental primer or dental adhesive.

The dental primers and dental adhesives of the invention may includeone, two, three, four or more antibacterial agents of differentidentity. As non-limiting examples, the dental primers may include:

(a) one type of antibacterial monomer;(b) one type of silver-containing nanoparticle;(c) one type of quaternary ammonium salt;(d) one type of antibacterial monomer and one type of silver-containingnanoparticle;(e) one type of antibacterial monomer and one type of quaternaryammonium salt;(f) one type of silver-containing nanoparticle and one type ofquaternary ammonium salt;(g) one type of antibacterial monomer, one type of silver-containingnanoparticle, and one type of quaternary ammonium salt;(h) two types of antibacterial monomer and one type of silver-containingnanoparticle;(i) two types of antibacterial monomer and one type of quaternaryammonium salt; or(j) two types of antibacterial monomer, one type of silver-containingnanoparticle, and one type of quaternary ammonium salt.

As non-limiting examples, the dental adhesives may include:

(a) one type of antibacterial monomer;(b) one type of silver-containing nanoparticle;(c) one type of quaternary ammonium salt;(d) one type of antibacterial monomer and one type of silver-containingnanoparticle;(e) one type of antibacterial monomer and one type of quaternaryammonium salt;(f) one type of silver-containing nanoparticle and one type ofquaternary ammonium salt;(g) one type of antibacterial monomer, one type of silver-containingnanoparticle, and one type of quaternary ammonium salt;(h) two types of antibacterial monomer and one type of silver-containingnanoparticle;(i) two types of antibacterial monomer and one type of quaternaryammonium salt;(j) two types of antibacterial monomer, one type of silver-containingnanoparticle, and one type of quaternary ammonium salt;(k) one type of antibacterial monomer and NACP;(l) one type of silver-containing nanoparticle and NACP;(m) one type of quaternary ammonium salt and NACP;(n) one type of antibacterial monomer, one type of silver-containingnanoparticle and NACP;(o) one type of antibacterial monomer, one type of quaternary ammoniumsalt and NACP;(p) one type of silver-containing nanoparticle, one type of quaternaryammonium salt and NACP;(q) one type of antibacterial monomer, one type of silver-containingnanoparticle, one type of quaternary ammonium salt and NACP;(r) two types of antibacterial monomer, one type of silver-containingnanoparticle and NACP;(s) two types of antibacterial monomer, one type of quaternary ammoniumsalt and NACP; or(t) two types of antibacterial monomer, one type of silver-containingnanoparticle, one type of quaternary ammonium salt and NACP.

Non-limiting examples of the dental primers of the present inventioninclude:

(a) a dental primer comprising about 95% primer and about 5% QADM bymass;(b) a dental primer comprising about 90% primer and about 10% QADM bymass;(c) a dental primer comprising about 99.95% primer and about 0.05% NAgby mass;(d) a dental primer comprising about 99.9% primer and about 0.1% NAg bymass;(e) a dental primer comprising about 89.95% primer, about 10% QADM andabout 0.05% NAg by mass;(f) a dental primer comprising about 95% primer and about 5% DMAHM bymass;(g) a dental primer comprising about 95% primer and about 5% DMADDM bymass;(h) a dental primer comprising about 94.9% primer, about 5% DMAHM bymass and about 0.1% NAg by mass; or(i) a dental primer comprising about 94.9% primer, about 5% DMADDM bymass and about 0.1% NAg by mass. In each of these examples, the primerin the dental primer may be any of the primers disclosed here, including(a) SBMP primer comprising 35-45% 2-hydroxyethylmethacrylate (HEMA),10-20% copolymer of acrylic and itaconic acids, and 40-50% water; and(b) PMGDM/HEMA at 3.3/1 ratio+1% BAPO+50% acetone.

Non-limiting examples of the dental adhesives of the present inventioninclude:

(a) a dental adhesive comprising about 90% adhesive and about 10% QADMby mass;(b) a dental adhesive comprising about 99.95% adhesive and about 0.05%NAg by mass;(c) a dental adhesive comprising about 99.9% adhesive and about 0.1% NAgby mass;(d) a dental adhesive comprising about 89.95% adhesive, about 10% QADMand about 0.05% NAg by mass;(e) a dental adhesive comprising about 89.9% adhesive, about 10% QADMand about 0.1% NAg by mass;(f) a dental adhesive comprising about 89.9% adhesive, about 10% NACPand about 0.1% NAg by mass;(g) a dental adhesive comprising about 79.9% adhesive, about 20% NACPand about 0.1% NAg by mass;(h) a dental adhesive comprising about 69.9% adhesive, about 30% NACPand about 0.1% NAg by mass;(i) a dental adhesive comprising about 59.9% adhesive, about 40% NACPand about 0.1% NAg by mass;(j) a dental adhesive comprising about 79.9% adhesive, about 10% NACP,about 10% QADM and about 0.1% NAg by mass;(k) a dental adhesive comprising about 59.9% adhesive, about 20% NACP,about 20% QADM and about 0.1% NAg by mass;(l) a dental adhesive comprising about 59.9% adhesive, about 30% NACP,about 10% QADM and about 0.1% NAg by mass;(m) a dental adhesive comprising about 49.9% adhesive, about 40% NACP,about 10% QADM and about 0.1% NAg by mass;(n) a dental adhesive comprising about 95% adhesive and about 5% DMAHMby mass;(o) a dental adhesive comprising about 95% adhesive and about 5% DMADDMby mass;(p) a dental adhesive comprising about 94.9% adhesive, about 5% DMAHM bymass and about 0.1% NAg by mass; or(q) a dental adhesive comprising about 94.9% adhesive, about 5% DMADDMby mass and about 0.1% NAg by mass. In each of these example, theadhesive in the dental adhesive may be any of the adhesives disclosedhere, including (a) SBMP adhesive comprising 60-70% BisGMA and 30-40%HEMA; and (b) BisGMA/TEGMA at 7/3 ratio+1% BAPO.

The dental bonding systems of the invention include a two-componentdental bonding system comprising a dental primer and a dental adhesive,a three-step bonding system, a two-step bonding system, and a one-stepself-adhesive bonding system.

The two-component dental bonding systems of the invention comprise (i) adental primer and (ii) a dental adhesive, wherein one or both the dentalprimer and the dental adhesive has antibacterial properties. In arelated embodiment, the two-component dental bonding systems comprise(i) a dental primer and (ii) a dental adhesive, wherein one or both thedental primer and the dental adhesive has antibacterial properties, andwherein one or both the dental primer and the dental adhesive hasremineralizing properties. The two-component dental bonding system maybe used, in one example, as follows. Before using the bonding system,the tooth is prepared by removing decayed material, cleaning theenamel/dentin surface and applying an etchant to the enamel/dentinsurface. A bonding system is then utilized, which can comprise, as anexample, application of a dental primer, followed by a dental adhesive.After curing the adhesive, a composite is packed into the void of thetooth. In some aspects, the dental primer or the dental adhesive in thetwo-component dental bonding systems may exclude antibacterial agents.

Three-step dental bonding systems of the invention include those thatcomprise (i) an etchant, (ii) a dental primer, and (iii) a dentaladhesive, wherein one or both the dental primer and the dental adhesivehas antibacterial properties. In certain aspects, one or both of thedental primer and the dental adhesive also has remineralizingproperties.

Two-step dental bonding systems of the invention include those thatcomprise (i) an etchant, and (ii) a mixture comprising a dental primerand a dental adhesive, wherein one or both the dental primer and thedental adhesive has antibacterial properties. In certain aspects, one orboth of the dental primer and the dental adhesive also hasremineralizing properties.

One-step self-adhesive bonding systems of the invention include thosethat comprise a mixture that comprises an etchant, a dental primer, anda dental adhesive, wherein one or both the dental primer and the dentaladhesive has antibacterial properties. In certain aspects, one or bothof the dental primer and the dental adhesive also has remineralizingproperties.

Suitable etchants for use in the dental bonding systems of the presentinvention include Scotchbond Multi-Purpose (SBMP) (3M, St. Paul, Minn.)etchant which contains 37% phosphoric acid.

Each of the dental bonding systems, dental primers and dental adhesivesof the present invention are suitable for use in the teeth of mammals,including primates such as human or non-human primates, and those ofdogs, cats, horses, cattle, pigs, goats and sheep, for example.

The dental bonding systems, dental primers and dental adhesivesdescribed herein can be used in a method of inhibiting growth ofaciduric bacteria on a surface of a tooth of a subject, comprisingrestoring a surface of the tooth from which a decayed portion has beenremoved by applying a dental composite using a dental bonding system,dental primer and/or dental adhesive described herein to the surface ofthe tooth, thereby inhibiting growth of aciduric bacteria on the toothof the subject.

The dental bonding systems, dental primers and dental adhesivesdescribed herein can also be used in a method of inhibiting furtherdecay of a decaying tooth in a subject, comprising restoring a surfaceof the tooth from which a decayed portion has been removed by applying adental composite using a dental bonding system, dental primer and/ordental adhesive described herein to the surface of the tooth, therebyinhibiting further decay of the decaying tooth in the subject.

EXAMPLES Example 1: Antibacterial Dental Primers with QuaternaryAmmonium and Silver Nanoparticles

The objective of this study was to develop antibacterial dental primersby incorporation of QADM and NAg, and to investigate biofilm viabilityand acid production using a dental plaque microcosm model. It was foundthat: (1) Incorporating QADM or NAg into a dental primer did notcompromise dentin shear bond strength; (2) QADM-NAg-containing dentalprimer, in uncured or cured state, was strongly antibacterial; (3)Combining QADM and NAg together in a dental primer yielded a strongerantibacterial capability than each agent alone.

Materials and Methods

SCOTCHBOND MULTI-PURPOSE™ adhesive (referred as “SBMP”) (3M, St. Paul,Minn.) was used as the parent adhesive to test the effect of QADM-NAgincorporation. SBMP etchant contained 37% phosphoric acid. SBMP primercontained 35-45% 2-hydroxyethylmethacrylate (HEMA), 10-20% copolymer ofacrylic/itaconic acids, and 40-50% water. SBMP adhesive contained 60-70%BisGMA and 30-40% HEMA. QADM-NAg were incorporated into SBMP primer.SBMP etchant and adhesive were not modified.

The QADM, bis(2-methacryloyloxyethyl) dimethylammonium bromide, wasrecently synthesized (Antonucci et al., 2011; Cheng et al., 2012a).Briefly, 10 mmol of 2-(N,N-dimethylamino)ethyl methacrylate (DMAEMA,Sigma-Aldrich, St. Louis, Mo.) and 10 mmol of 2-bromoethyl methacrylate(BEMA, Monomer-Polymer Labs, Trevose, Pa.) were combined with 3 g ofethanol, and stirred at 60° C. for 24 h. The solvent was thenevaporated, yielding QADM. QADM was mixed with SBMP primer atQADM/(primer+QADM)=10% by mass, following a previous study (Cheng etal., 2012a).

Silver 2-ethylhexanoate (Strem, New Buryport, Mass.) of 0.08 g wasdissolved into 1 g of 2-(tert-butylamino)ethyl methacrylate (TBAEMA,Sigma). TBAEMA could facilitate Ag salt dissolution in the resin (ChengY J et al., 2011). This Ag solution was mixed with SBMP primer at 0.05%by mass of silver 2-ethylhexanoate (Cheng et al., 2012a).

Hence, the following four primers were tested: (1) SBMP primer(control); (2) control primer+10% QADM (termed “10QADM”); (3) controlprimer+0.05% NAg (termed “0.05NAg”); (4) control primer+10% QADM+0.05%NAg (termed “10QADM+0.05NAg”).

A transmission electron microscope (TEM, Tecnai-T12, FEI Company,Hillsboro, Oreg.) was used to examine the silver nanoparticles in theresin. Following a previous study (Cheng Y J et al., 2011), a thin sheetof mica was partially-split and the Ag-containing resin was placed inthe gap. The resin in the mica was pressed to form a film and thenphoto-cured. The resin film was used for TEM examination.

Extracted caries-free human third molars were sawed to remove the crowns(Isomet, Buehler, Lake Bluff, Ill.). The mid-coronal dentin surface wasground perpendicular to the longitudinal axis of the tooth on 320-gritSiC paper until occlusal enamel was completely removed. The dentinsurface was etched with 37% phosphoric acid for 15 s and rinsed withwater (Antonucci et al., 2009). A primer was applied, and the solventwas removed with an air stream. The adhesive was applied and light-curedfor 10 s (Optilux-VCL401, Demetron, Danbury, Conn.).

A stainless-steel iris having a central opening (diameter=4 mm,thickness=1.5 mm) was held against the adhesive-treated dentin(Antonucci et al., 2009). The central opening was filled with acomposite (TPH, Caulk/Dentsply, Milford, Del.) and light-cured for 60 s.Specimens were stored in water at 37° C. for 24 h. Dentin shear bondstrength, S_(D), was measured following a previous method (Antonucci etal., 2009). A chisel was aligned parallel to the composite-dentininterface, and loaded via a computer-controlled Universal TestingMachine (MTS, Eden Prairie, Minn.) at 0.5 mm/min until thecomposite-dentin bond failed. S_(D)=4P/(πd²), where P is load atfailure, and d is composite diameter.

Saliva was collected from a healthy adult donor having natural dentitionwithout active caries or periopathology, and without using antibioticswithin the last 3 months. The donor did not brush teeth for 24 h andabstained from food/drink intake for 2 h prior to donating saliva.Stimulated saliva was collected during parafilm chewing and kept on ice.The saliva was diluted in sterile glycerol to a concentration of 30%(Cheng L et al., 2011).

The un-cured primers were tested in agar disk diffusion. Thesaliva-glycerol was added to a growth medium containing mucin(concentration=2.5 g/L), bacteriological peptone (concentration=2.0g/L), tryptone (concentration=2.0 g/L), yeast extract (concentration=1.0g/L), NaCl (concentration=0.35 g/L), KCl (concentration=0.2 g/L), CaCl₂(concentration=0.2 g/L), and cysteine hydrochloride (concentration=0.1g/L) (pH=7.0) (McBain, 2009). The inoculum was incubated (37° C., 5%CO₂) for 24 h, and then used for the agar disk diffusion test. Itemployed three types of media. First, tryptic soy blood agar plates wereused to determine total microorganisms (Cheng L et al., 2011). Second,mitis salivarius agar (MSA) plates, containing 15% sucrose, were used todetermine total streptococci. Third, MSA plates plus 0.2 units ofbacitracin/mL was used to determine mutans streptococci.

Bacteria suspension of 0.4 mL was poured onto each agar plate(diameter=90 mm, thickness=4 mm). Then, 30 μL of each primer wasimpregnated into a sterile paper disk (diameter=9 mm, thickness=1.5 mm)(Imazato et al., 2006). The primer-impregnated paper disk (referred toas “disk”) was placed on a plate with bacteria, and incubated for 48 h.Bacteria inhibition zone size=(Outer diameter of inhibition zone−diskdiameter)/2.

Cured specimens were fabricated as schematically-shown in FIG. 24A,following a previous study (Imazato et al., 1998). A polyethylene mold(inner diameter=9 mm, thickness=2 mm) was situated on a glass slide.Each primer was brushed into the mold on the glass slide. After dryingwith a stream of air, SBMP adhesive was applied and cured for 20 s withOptilux. Then, a composite (TPH) was placed on the adhesive to fill themold and light-cured for 1 min. Specimens were agitated in water for 1 hto remove uncured monomers (Imazato et al., 1998).

Each specimen was placed into a well of 24-well plates with the primerfacing up. 1.5 mL of inoculum was added to each well. The assemblieswere incubated for 8 h. The specimens were then transferred to new24-well plates with fresh medium. After 16 h, the specimens weretransferred to new 24-well plates with fresh medium and incubated for 24h (Cheng L et al., 2011). Then, the biofilms on specimens were washedwith phosphate-buffered saline (PBS), live/dead stained (MolecularProbes, Eugene, Oreg.), and examined using an epifluorescence microscope(TE2000-S, Nikon, Melville, N.Y.).

For colony-forming unit (CFU) counts, specimens with 2-d biofilm wererinsed with cysteine peptone water (CPW) to remove loose bacteria. Thespecimens were transferred to 24-well plates containing buffered-peptonewater (BPW) plus 0.2% sucrose, and incubated for 3 h to allow biofilmsto produce acid. Lactate concentrations were determined using anenzymatic method (Cheng L et al., 2011). A microplate reader(SpectraMax, Molecular Devices, Sunnyvale, Calif.) was used to measurethe 340-nm absorbance. Standard curves were prepared using a lactic acidstandard (Supelco, Bellefonte, Pa.).

Specimens were transferred into tubes with 2-mL CPW, and the biofilmswere harvested by sonication/vortexing (Fisher, Pittsburgh, Pa.). Threetypes of agar plates were used: Tryptic soy blood plates to determinetotal microorganisms; MSA plus 15% sucrose to determine totalstreptococci; and MSA plus 0.2 units of bacitracin/mL to determinemutans streptococci.

One-way and two-way analyses-of-variance (ANOVA) were performed todetect the significant effects of variables. Tukey's multiple comparisonwas used to compare the data at p=0.05.

Results

TEM examination showed NAg particle sizes ranging 2-5 nm (FIG. 1A).Measurement of 100 particles (mean±sd) yielded (2.7±0.6) nm. The NAgwere well-dispersed in the resin, without noticeable agglomeration.Dentin shear bond strengths (FIG. 1B) were about 30-35 MPa. Adding QADMand NAg into primer did not compromise the bond strength (p>0.1).

Un-cured QADM-NAg primers had a strong antibacterial activity (FIG. 2).Control primer had a minimal inhibition zone (A). Primers 10QADM (B),0.05NAg (C), and 10QADM+0.05NAg (D) had much larger inhibition zones.Inhibition zone sizes are plotted in (E-G) for total microorganisms,total streptococci, and mutans streptococci, respectively. Inhibitionzone sizes for 10QADM+0.05NAg were 8-9 fold those of control (p<0.05).

Biofilms were grown on cured primers (FIG. 24A). Control primer had fullcoverage of primarily live bacteria, while 10QADM, 0.05NAg, and10QADM+0.05NAg exhibited strong antibacterial activity. The 10QADM,0.05NAg, and 10QADM+0.05NAg primers greatly reduced the CFU counts (FIG.3A-C). CFU on 10QADM+0.05NAg was approximately an order of magnitudeless than those on control primer. Lactic acid from biofilms (FIG. 3D)was reduced on 10QADM, 0.05NAg, and 10QADM+0.05NAg, compared to control(p<0.05). These results demonstrate that adding QADM or NAg into primerimparted a potent antibacterial effect, and the strongest effect wasachieved by combining QADM and NAg in the primer.

Example 2: Effect of Quaternary Ammonium and SilverNanoparticle-Containing Adhesives on Dentin Bond Strength and DentalPlaque Microcosm Biofilms

The objective of this study were to develop an antibacterial,two-component, dental bonding system by incorporation of quaternaryammonium dimethacrylate (QADM) and nanoparticles of silver (NAg) intodental primers and dental adhesives, and to investigate the effect ofQADM-NAg-containing adhesive and primer on dentin bond strength andplaque microcosm biofilm response for the first time. Adding QADM andNAg into adhesive and primer did not compromise the dentin shear bondstrength which ranged from 30 to 35 MPa (p>0.1). Adding QADM or NAgmarkedly reduced the biofilm viability, compared to adhesive control.QADM and NAg together in the adhesive had a much stronger antibacterialeffect than using each agent alone (p<0.05). Adding QADM and NAg in bothadhesive and primer had the strongest antibacterial activity, reducingmetabolic activity, CFU, and lactic acid by an order of magnitude,compared to control.

Materials and Methods QADM Incorporation

Scotchbond Multi-Purpose bonding system (3M, St. Paul, Minn.), referredas “SBMP”, was used as the parent bonding system to test the effect ofincorporation of QADM and NAg. The purpose was to investigate a modelsystem, and then the method of QADM and NAg incorporation could beapplied to other adhesive systems. According to the manufacturer, SBMPetchant contains 37% phosphoric acid. SBMP primer single bottle contains35-45% 2-Hydroxyethylmethacrylate (HEMA), 10-20% copolymer of acrylicand itaconic acids, and 40-50% water. SBMP adhesive contains 60-70%BisGMA and 30-40% HEMA.

Bis(2-methacryloyloxyethyl) dimethylammonium bromide was a quaternaryammonium dimethacrylate (QADM), and was recently synthesized andincorporated into dental composites (Antonucci et al. 2011; Cheng L. etal. 2011). The synthesis of QADM was performed using a modifiedMenschutkin reaction, where a tertiary amine group was reacted with anorgano-halide. A benefit of this reaction is that the reaction productsare generated at virtually quantitative amounts and require minimalpurification (Antonucci et al. 2011). Briefly, 10 mmol of2-(N,N-dimethylamino)ethyl methacrylate (DMAEMA, Sigma, St. Louis, Mo.)and 10 mmol of 2-bromoethyl methacrylate (BEMA, Monomer-Polymer andDajec Labs, Trevose, Pa.) were combined with 3 g of ethanol in a 20 mLscintillation vial. The vial was stirred at 60° C. for 24 h. The solventwas then removed, yielding QADM as a clear, colorless, and viscousliquid. The QADM was mixed with the SBMP adhesive or primer at a QADMmass fraction of 10%. QADM mass fractions of 20% or higher were not useddue to a decrease in dentin bond strength in preliminary study.

NAg Incorporation

Silver 2-ethylhexanoate powder (Strem, New Buryport, Mass.) wasdissolved in 2-(tert-butylamino)ethyl methacrylate (TBAEMA, Sigma) at0.08 g of silver salt per 1 g of TBAEMA, following previous studies(Cheng L. et al. 2011; Cheng Y J. et al. 2011). TBAEMA was used becauseit improves the solubility by forming Ag—N coordination bonds with Agions, thereby facilitating the Ag salt to dissolve in the resinsolution. TBAEMA was selected since it contains reactive methacrylategroups and therefore can be chemically incorporated into a dental resinupon photopolymerization (Cheng L. et al. 2011; Cheng Y J. et al. 2011).This method produced NAg with a mean particle size of 2.7 nm that werewell dispersed in the resin matrix (Cheng L. et al. 2011). The Agsolution was mixed with SBMP adhesive at silver 2-ethylhexanoate massfractions of 0.05% and 0.1%. Ag mass fractions of 0.15% or higher werenot used due to a decrease in dentin bond strength.

Dentin Shear Bond Testing and SEM Examination

As listed in Table 1, six groups were used for dentin shear bondstrength testing. The purpose of groups 1-3 was to investigate theeffects of QADM or NAg individually. The purpose of 3 and 4 was toexamine the effect of NAg mass fraction. The purpose of comparing 2, 3and 5 was to examine the effect of combining QADM and NAg together inthe same adhesive. The purpose of comparing 5 with 6 was to investigatethe effects of adding QADM and NAg into both the adhesive and the primeron dentin bond strength and biofilm response.

TABLE 1 Compositions of adhesive resin and dentin primer* Group Adhesiveresin Dentin primer Group name 1 Control Control Control 2 Control + 10%QADM Control A + 10QADM 3 Control + 0.05% NAg Control A + 0.05NAg 4Control + 0.1% NAg Control A + 0.1NAg 5 Control + 10% QADM + Control A +10QADM + 0.05% NAg 0.05NAg 6 Control + 10% QADM + Control + A&P +10QADM + 0.05% NAg 10% QADM + 0.05NAg 0.05% NAg *The control adhesivewas the SBMP adhesive without modification. The control primer was theSBMP primer without modification. QADM = quaternary ammoniumdimethacrylate. NAg = nanoparticles of silver. In the “Group name”column, A = adhesive resin. P = primer.

Extracted caries-free human third molars were cleaned and stored in0.01% thymol solution. Flat mid-coronal dentin surfaces were prepared bycutting off the tips of molar crowns with a diamond saw (Isomet,Buehler, Lake Bluff, Ill.). Each tooth was embedded in a poly-carbonateholder (Bosworth, Skokie, Ill.) and ground perpendicular to thelongitudinal axis on 320-grit silicon carbide paper until the occlusalenamel was completely removed. As shown schematically in FIG. 4A, thedentin surface was etched with 37% phosphoric acid gel for 15 s andrinsed with distilled water for 15 s, following a previous study(Antonucci et al. 2009). The primer was applied with a brush-tippedapplicator and rubbed in for 15 s. The solvent was removed with a streamof air for 5 s. Then the adhesive was applied and light-cured for 10 s(Optilux VCL 401, Demetron Kerr, Danbury, Conn.). A stainless-steeliris, having a central opening with a diameter of 4 mm and a thicknessof 1.5 mm, was held against the adhesive-treated dentin surface. Thecentral opening was filled with a composite (TPH, Caulk/Dentsply,Milford, Del.), and light-cured for 60 s. The bonded specimens werestored in distilled water at 37° C. for 24 h.

The dentin shear bond strength, S_(D), was measured as shownschematically in FIG. 4B (Antonucci et al. 2009). The chisel wasconnected with a computer-controlled Universal Testing Machine (MTS,Eden Prairie, Minn.) and held parallel to the composite-dentininterface. Load was applied at a rate of 0.5 mm/min until the bondfailed. S_(D) was calculated as: S_(D)=4P/(πd²), where P is the load atfailure, and d is the diameter of the composite. Ten teeth were testedfor each group (n=10).

The bonded tooth was cut through the center in the longitudinaldirection via the diamond saw (Isomet) with copious water. Threespecimens were prepared for each group. The sectioned surface waspolished with increasingly finer SiC paper up to 4000 grit. Following aprevious study (Imazato et al. 2003), the polished surface was treatedwith 50% phosphoric acid for 30 s, then with 10% NaOCl for 2 min. Afterbeing thoroughly rinsed with water for 10 min, the specimens were airdried and then sputter-coated with gold. The dentin-adhesive bondedinterfaces were then examined via scanning electron microscopy (SEM,Quanta 200, FEI, Hillsboro, Oreg.).

Saliva Collection for Plaque Microcosm Model

The dental plaque microcosm model was approved by the University ofMaryland. Human saliva was shown to be ideal for growing plaquemicrocosm biofilms in vitro, with the advantage of maintaining much ofthe complexity and heterogeneity of the dental plaque in vivo (McBain AJ. 2009). The saliva for biofilm inoculums was collected from a healthyadult donor having natural dentition without active caries orperiopathology, and without the use of antibiotics within the last 3months, following a previous study (Cheng L. et al. 2011). The donor didnot brush teeth for 24 h and abstained from food/drink intake for atleast 2 h prior to donating saliva. Stimulated saliva was collectedduring parafilm chewing and kept on ice. The saliva was diluted insterile glycerol to a concentration of 30%, and stored at −80° C. (ChengL. et al. 2011).

Specimen Fabrication for Biofilm Experiments

Layered disk specimens for biofilm experiments were fabricated followingprevious studies (Imazato et al. 1998; Li et al. 2009). A polyethylenedisk mold (inner diameter=9 mm, thickness=2 mm) was situated on a glassslide. For groups 1-5, each adhesive was applied into the mold to coverthe glass slide. Then, a composite (TPH) was placed onto the adhesive tofill the disk mold and light-cured for 1 min. For group 6, the primerwas first applied into the mold to cover the glass slide. After dryingwith a stream of air, the adhesive was applied and cured for 20 s withOptilux. Then, a composite (TPH) was placed on the adhesive to fill thedisk mold and light-cured for 1 min. The disks were immersed in sterilewater and agitated for 1 h to remove any uncured monomer, following aprevious study (Imazato et al. 1998). The disks were then dried andsterilized with ethylene oxide (Anprolene AN 74i, Andersen, Haw River,N.C.).

Six groups were tested in biofilm experiments. Groups 1-5 had specimenswith adhesives 1-5 (Table 1) covering the top surface of the compositedisk, without primer, in order to test the antibacterial properties ofthe adhesives, as shown schematically in FIG. 24B. Group 6 had theQADM-NAg primer covering the adhesive on the composite disk in order totest the antibacterial properties of the primer/adhesive combination, asshown schematically in FIG. 24A.

MTT Assay of Metabolic Activity

The saliva-glycerol stock was added, with 1:50 final dilution, to agrowth medium as inoculum. The growth medium contained mucin (type II,porcine, gastric) at a concentration of 2.5 g/L; bacteriologicalpeptone, 2.0 g/L; tryptone, 2.0 g/L; yeast extract, 1.0 g/L; NaCl, 0.35g/L, KCl, 0.2 g/L; CaCl₂, 0.2 g/L; cysteine hydrochloride, 0.1 g/L;haemin, 0.001 g/L; vitamin K1, 0.0002 g/L, at pH 7 (McBain et al. 2005).The inoculum was cultured at 37° C. in an incubator containing 5% CO₂for 24 h. Each disk specimen was placed into a well of 24-well plates,with the antibacterial surface on the top. 1.5 mL of inoculum was addedto each well, and incubated in 5% CO₂ at 37° C. for 8 h. The disks werethen transferred to new 24-well plates with fresh medium and incubated.After 16 h, the disks were transferred to new 24-well plates with freshmedium and incubated for 24 h. This 2-day (d) incubation formed plaquemicrocosm biofilms as shown previously (Cheng L. et al. 2011).

The MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)assay is a colorimetric assay that measures the enzymatic reduction ofMTT, a yellow tetrazole, to formazan (Antonucci et al. 2011; Cheng L. etal. 2011). Each disk with the 2-d biofilm was transferred to a new24-well plate, then 1 mL of MTT dye (0.5 mg/mL MTT in PBS) was added toeach well and incubated at 37° C. in 5% CO₂ for 1 h. During thisprocess, metabolically active bacteria reduced the MTT to purpleformazan. After 1 h, the disks were transferred to a new 24-well plate,1 mL of dimethyl sulfoxide (DMSO) was added to solubilize the formazancrystals, and the plate was incubated for 20 min with gentle mixing atroom temperature in the dark. After mixing via pipetting, 200 μL of theDMSO solution from each well was transferred to a 96-well plate, and theabsorbance at 540 nm (optical density OD540) was measured via amicroplate reader (SpectraMax M5, Molecular Devices, Sunnvale, Calif.).A higher absorbance is related to a higher formazan concentration, whichindicates a higher metabolic activity in the biofilm on the disk.

Lactic Acid Production and Colony-Forming Unit (CFU) Counts

Each disk with the 2-d biofilm was rinsed with cysteine peptone water(CPW) to remove loose bacteria. The disks were transferred to 24-wellplates containing buffered peptone water (BPW) plus 0.2% sucrose. Thesamples were incubated in 5% CO₂ at 37° C. for 3 h to allow the biofilmsto produce acid. The BPW solutions were then stored for lactateanalysis.

Disks with biofilms were transferred into tubes with 2 mL CPW, and thebiofilms were harvested by sonication and vortexing via a vortex mixer(Fisher, Pittsburgh, Pa.). Three types of agar plates were used. First,tryptic soy blood agar culture plates were used to determine totalmicroorganisms (Cheng L. et al. 2011). Second, mitis salivarius agar(MSA) culture plates, containing 15% sucrose, were used to determinetotal streptococci (Lima et al. 2009). This is because MSA containsselective agents crystal violet, potassium tellurite and trypan blue,which inhibit most gram-negative bacilli and most gram-positive bacteriaexcept streptococci, thus enabling streptococci to grow (Lima et al.2009). Third, cariogenic mutans streptococci are known to be resistantto bacitracin, and this property is often used to isolate mutansstreptococci from the highly heterogeneous oral microflora. Hence, MSAagar culture plates plus 0.2 units of bacitracin per mL was used todetermine mutans streptococci (Hildebrandt et al. 2006).

Lactate concentrations in the BPW solutions were determined using anenzymatic (lactate dehydrogenase) method, following a previous study(Cheng L. et al. 2011). The microplate reader was used to measure theabsorbance at 340 nm (optical density OD₃₄₀) for the collected BPWsolutions. Standard curves were prepared using a lactic acid standard(Supelco, Bellefonte, Pa.).

One-way analysis of variance (ANOVA) was performed to detect thesignificant effects of the variables. Tukey's multiple comparison wasused to compare the data at a p value of 0.05.

Results

FIG. 4 shows schematics of the dentin shear bond test and the strengthresults: (A) Schematic of specimen preparation, (B) schematic of shearbond testing, (C) dentin shear bond strength results. In (C), each valueis mean±sd (n=10). The six groups had shear bond strengths that were notsignificantly different (p>0.1), indicating that adding QADM and NAg toadhesive and primer did not compromise the dentin shear bond strength.

The dentin-adhesive interfaces were examined via SEM, and representativeimages are shown in FIG. 5: (A) SBMP control, and (B)A&P+10QADM+0.05NAg. “HL” refers to the hybrid layer between the adhesiveand the underlying mineralized dentin. “T” indicates the resin tagsformed by the adhesive resin filling into the dentinal tubules. Numerousresin tags were found in samples of all six groups. The short arrow in(A) indicates a short resin tag. The long arrow indicates an example ofa long resin tag. Some tags were shorter because, during samplepreparation, the sectioning surface was not exactly parallel to the longaxis of dentinal tubules. Some tubules were intersected by the cuttingand thus shortened. A mixture of long and short tags was observed in allsamples. There was no noticeable difference between the six groups.

FIG. 6 shows metabolic activity. Groups 1-5 followed schematic of FIG.24B. Group 6 used the QADM-NAg primer and followed schematic of FIG.24A. Biofilms on the as-received commercial adhesive had a highmetabolic activity. Incorporation of QADM and NAg each markedly reducedthe metabolic activity (p<0.05). Adding QADM and NAg together in theadhesive resulted in a much lower metabolic activity than using QADM orNAg alone (p<0.05). Adding QADM and NAg both in the primer and in theadhesive yielded the lowest biofilm metabolic activity (p<0.05). Themetabolic activity of biofilms on A&P+10QADM+0.05NAg was nearly an orderof magnitude less than that on adhesive control.

FIG. 7 plots CFU counts for: (A) Total microorganisms, (B) totalstreptococci, and (C) mutans streptococci. Adding QADM or NAg eachdecreased the CFU, compared to the as-received commercial adhesivecontrol (p<0.01). Increasing the NAg mass fraction from 0.05% to 0.1%decreased the CFU (p<0.05). The combination of QADM and NAg in theadhesive had a much stronger antibacterial effect than using QADM or NAgalone (p<0.05). Incorporation of QADM and NAg together, in both theprimer and the adhesive, had the greatest reductions in CFU (p<0.05).All three CFU counts for biofilms on A&P+10QADM+0.05NAg were reduced byan order of magnitude, compared to those on the as-received commercialadhesive.

FIG. 8 plots the lactic acid production by biofilms. Biofilms onadhesive control produced the most acid. Adding QADM or NAg eachdecreased the acid production (p<0.05). Adhesive with 0.1% NAg had lessacid than that with 0.05% NAg (p<0.05). Adhesive with both 10% QADM and0.05% NAg had less acid than those using either 10% QADM or 0.05% NAg(p<0.05). When 10% QADM and 0.05% NAg were incorporated into both theadhesive resin and the primer, the lactic acid production was furtherreduced (p<0.05).

Example 3: Antibacterial Dental Adhesive Containing Silver and AmorphousCalcium Phosphate Nanoparticles

The objectives of this study were to incorporate nanoparticles of silver(NAg) and nanoparticles of amorphous calcium phosphate (NACP) into adental adhesive, and to investigate the effects on dentin bond strengthand dental plaque microcosm biofilm response. Human dentin shear bondstrengths ranged from 26 to 34 MPa; adding NAg and NACP did notsignificantly decrease the bond strength (p>0.1). Bonding agentscontaining NAg and NACP greatly reduced the biofilm viability andmetabolic activity, compared to the control. CFU for totalmicroorganisms, total streptococci, and mutans streptococci on bondingagents with NACP and NAg were an order of magnitude less than those ofcontrol. Lactic acid production for groups containing NACP and NAg werereduced to ¼ of that of control. Microcosm biofilm viability and acidproduction were greatly reduced on bonding agents containing NAg andNACP nanoparticles for the first time, without compromising dentin bondstrength.

Materials and Methods

NAg Incorporation into Primer and Adhesive

Scotchbond Multi-Purpose (3M, St. Paul, Minn.), referred to as “SBMP”,was used as the parent bonding system to test the effect ofincorporation of NACP and NAg. The purpose was to investigate a modelsystem, and then the method of incorporating NACP and NAg could beapplied to other bonding agents. According to the manufacturer, SBMPetchant contained 37% phosphoric acid. SBMP primer single bottlecontained 35-45% 2-Hydroxyethylmethacrylate (HEMA), 10-20% copolymer ofacrylic and itaconic acids, and 40-50% water. SBMP adhesive contained60-70% BisGMA and 30-40% HEMA.

Silver 2-ethylhexanoate powder (Strem, New Buryport, Mass.) wasdissolved in 2-(tert-butylamino)ethyl methacrylate (TBAEMA, Sigma) at0.08 g of silver salt per 1 g of TBAEMA, following previous studies(Cheng Y J. et al. 2011; Cheng L. et al. 2011). TBAEMA was used becauseit improves the solubility by forming Ag—N coordination bonds with Agions, thereby facilitating the Ag salt to dissolve in the resinsolution. TBAEMA was selected since it contains reactive methacrylategroups and can be chemically incorporated into a resin uponphotopolymerization. This method produced NAg with a mean particle sizeof 2.7 nm that were well dispersed in the resin (Cheng Y J. et al. 2011;Cheng L. et al. 2011). To incorporate NAg into the primer, theaforementioned Ag-TBAEMA solution was mixed with the SBMP primer at asilver 2-ethylhexanoate/(primer+silver 2-ethylhexanoate) of 0.1% bymass; this mass fraction was selected based on previous studies (Cheng YJ. et al. 2011; Cheng L. et al. 2011). To incorporate NAg into theadhesive, the Ag-TBAEMA was mixed with the SBMP adhesive at 0.1% massfraction.

Addition of NACP into Adhesive

Nanoparticles of ACP (Ca₃[PO₄]₂) were synthesized using a spray-dryingtechnique as described previously (Xu et al. 2006; Chow et al. 2004).Briefly, calcium carbonate (CaCO₃, Fisher, Fair Lawn, N.J.) anddicalcium phosphate anhydrous (CaHPO₄, Baker Chemical, Phillipsburg,N.J.) were dissolved into an acetic acid solution to obtain final Ca andPO₄ ionic concentrations of 8 mmol/L and 5.333 mmol/L, respectively. TheCa/P molar ratio for the solution was 1.5, the same as that for ACP. Thesolution was sprayed into the heated chamber of the spray-dryingapparatus. The dried particles were collected via an electrostaticprecipitator (AirQuality, Minneapolis, Minn.), yielding NACP with a meanparticle size of 116 nm (Xu et al. 2011).

The NACP were mixed with the adhesive containing 0.1% silver2-ethylhexanoate. The NACP mass fractions in the adhesive were: 0%, 10%,20%, 30%, and 40%, following previous studies on NACP nanocomposites (Xuet al. 2011; Moreau et al. 2011).

Hence, six bonding agents were tested:

[1]. SBMP primer, SBMP adhesive (termed “SBMP control”).[2]. Primer+0.1% NAg, adhesive+0.1% NAg (termed “P&A+NAg”. P=primer,A=adhesive).[3]. Primer+0.1% NAg, adhesive+0.1% NAg+10% NACP (termed “P&A+NAg,A+10NACP”).[4]. Primer+0.1% NAg, adhesive+0.1% NAg+20% NACP (termed “P&A+NAg,A+20NACP”).[5]. Primer+0.1% NAg, adhesive+0.1% NAg+30% NACP (termed “P&A+NAg,A+30NACP”).[6]. Primer+0.1% NAg, adhesive+0.1% NAg+40% NACP (termed “P&A+NAg,A+40NACP”).

Dentin Shear Bond Strength Testing and SEM Examination

Extracted caries-free human third molars were cleaned and stored in0.01% thymol solution. The tips of the molar crowns were cut off via adiamond saw (Isomet, Buehler, Lake Bluff, Ill.) to yield flatmid-coronal dentin surfaces. Following a previous study (Antonucci etal. 2009), the tooth was embedded in a poly-carbonate holder (Bosworth,Skokie, Ill.) and ground perpendicular to the longitudinal axis using320-grit SiC paper until there was no occlusal enamel left. The bondingprocedures are shown in FIG. 4A. Briefly, the dentin surface was etchedwith 37% phosphoric acid gel for 15 s and rinsed with water for 15 s(Antonucci et al. 2009). The primer was applied with a brush-tippedapplicator and rubbed in for 15 s, and the solvent was removed with astream of air. The adhesive was then applied and photo-cured for 10 s(Optilux VCL 401, Demetron Kerr, Danbury, Conn.). Then, astainless-steel iris with a central opening (diameter=4 mm,thickness=1.5 mm) was held against the adhesive-treated dentin surface.The central opening was filled with a composite (TPH, Caulk/Dentsply,Milford, Del.) and photo-cured for 60 s.

The bonded specimens were stored in distilled water at 37° C. for 24 h(Antonucci et al. 2009). Then, the dentin shear bond strength, S_(D),was measured as schematically shown in FIG. 4B. The chisel was connectedwith a computer-controlled Universal Testing Machine (MTS, Eden Prairie,Minn.), held parallel to the composite-dentin interface, and loaded at0.5 mm/min until the bond failed. S_(D) was calculated as:S_(D)=4P/(πd²), where P is the load at failure, and d is the diameter ofthe composite. Ten teeth were tested for each group (n=10).

For scanning electron microscopy (SEM) examination, the bonded tooth wascut through the center parallel to the longitudinal axis via a diamondsaw (Isomet) with copious water. The sectioned surface was polished withincreasingly finer SiC paper up to 4000 grit. Following a previous study(Imazato et al. 2007), the polished surface was treated with 50%phosphoric acid for 30 s, then with 10% NaOCl for 2 min. After beingthoroughly rinsed with water for 10 min, the specimens were air driedand then sputter-coated with gold. Three specimens were prepared foreach group. The specimens were then examined in an SEM (Quanta 200, FEI,Hillsboro, Oreg.).

Specimen Fabrication for Biofilm Experiments

Following previous studies (Imazato et al. 1998; Li et al. 2009),layered disk specimens were made as shown in FIG. 3A (Example 3). Apolyethylene mold (inner diameter=9 mm, thickness=2 mm) was situated ona glass slide. A primer was applied into the mold to cover the glass.After drying with a stream of air, an adhesive was applied and cured for20 s with Optilux. A composite (TPH) was placed on the adhesive tocompletely fill the mold, and light-cured for 1 min. The bondedspecimens were immersed in water and agitated for 1 h to remove anyuncured monomer (Imazato et al. 1998). The disks were then dried andsterilized with ethylene oxide (Anprolene AN 74i, Andersen, Haw River,N.C.).

Dental Plaque Microcosm Model and Live/Dead Assay

Saliva is ideal for growing dental plaque microcosm biofilms in vitrowhich maintain much of the complexity and heterogeneity of dental plaquein vivo (McBain et al. 2009). The dental plaque microcosm model wasapproved by the University of Maryland. Saliva was collected from ahealthy adult donor having natural dentition without active caries orperiopathology, and without the use of antibiotics within the last 3months (Cheng L. et al. 2011). The donor did not brush teeth for 24 hand abstained from food/drink intake for at least 2 h prior to donatingsaliva. Stimulated saliva was collected during parafilm chewing and kepton ice. The saliva was diluted in sterile glycerol to a concentration of30%, and stored at −80° C. (Cheng L. et al. 2011).

The saliva-glycerol stock was added, with 1:50 final dilution, to agrowth medium as inoculum. The growth medium contained mucin (type II,porcine, gastric) at a concentration of 2.5 g/L; bacteriologicalpeptone, 2.0 g/L; tryptone, 2.0 g/L; yeast extract, 1.0 g/L; NaCl, 0.35g/L, KCl, 0.2 g/L; CaCl₂, 0.2 g/L; cysteine hydrochloride, 0.1 g/L;haemin, 0.001 g/L; vitamin K1, 0.0002 g/L, at pH 7 (McBain et al. 2005).The inoculum was cultured in an incubator (5% CO₂, 37° C.) for 24 h.Each disk was placed into a well of 24-well plates, with the primer onthe top. Then, 1.5 mL of inoculum was added to each well, and incubatedfor 8 h. The disks were transferred to new 24-well plates with freshmedium and incubated. After 16 h, the disks were transferred to new24-well plates with fresh medium and incubated for 24 h. This totaled 2d of incubation, which was shown in a previous study to be sufficient toform microcosm biofilms (Cheng L. et al. 2011).

Disks with 2-d biofilms were washed three times with phosphate bufferedsaline (PBS), and then stained using a live/dead bacterial kit(Molecular Probes, Eugene, Oreg.). Live bacteria were stained with Syto9 to produce a green fluorescence, and bacteria with compromisedmembranes were stained with propidium iodide to produce a redfluorescence. Specimens were examined with an epifluorescence microscope(TE2000-S, Nikon, Melville, N.Y.).

MTT Assay of Metabolic Activity

MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assaywas used to measure the metabolic activity of biofilms (Cheng Y J. etal. 2011; Antonucci et al. 2011). MTT is a colorimetric assay thatmeasures the enzymatic reduction of MTT, a yellow tetrazole, toformazan. Each disk with the 2-d biofilm was transferred to a new24-well plate. One mL of MTT dye (0.5 mg/mL MTT in PBS) was added toeach well and incubated for 1 h. During this process, metabolicallyactive bacteria reduced the MTT to purple formazan. After 1 h, the diskswere transferred to a new 24-well plate, 1 mL of dimethyl sulfoxide(DMSO) was added to solubilize the formazan crystals, and the plate wasincubated for 20 min in the dark. After mixing via pipetting, 200 μL ofthe DMSO solution from each well was transferred to a 96-well plate, andthe absorbance at 540 nm (optical density OD540) was measured via amicroplate reader (SpectraMax M5, Molecular Devices, Sunnvale, Calif.).A higher absorbance is related to a higher formazan concentration, whichindicates a higher metabolic activity in the biofilm adherent on thedisk.

Lactic Acid Production and Colony-Forming Unit (CFU) Counts

Disks with 2-d biofilms were rinsed with cysteine peptone water (CPW) toremove loose bacteria, and transferred to 24-well plates containingbuffered peptone water (BPW) plus 0.2% sucrose. The samples wereincubated for 3 h to allow the biofilms to produce acid. The BPWsolutions were then stored for lactate analysis. Lactate concentrationswere determined using an enzymatic (lactate dehydrogenase) method (ChengL. et al. 2011). The microplate reader was used to measure theabsorbance at 340 nm (optical density OD₃₄₀) for the collected BPWsolutions. Standard curves were prepared using a lactic acid standard(Supelco, Bellefonte, Pa.).

Disks with biofilms were transferred into tubes with 2 mL CPW, and thebiofilms were harvested by sonication and vortexing via a vortex mixer(Fisher, Pittsburgh, Pa.). Three types of agar plates were used. First,tryptic soy blood agar culture plates were used to determine totalmicroorganisms (Cheng L. et al. 2011). Second, mitis salivarius agar(MSA) culture plates, containing 15% sucrose, were used to determinetotal streptococci (Lima et al. 2009). This is because MSA containsselective agents crystal violet, potassium tellurite and trypan blue,which inhibit most gram-negative bacilli and gram-positive bacteriaexcept streptococci, thus enabling streptococci to grow (Lima et al.2009). Third, cariogenic mutans streptococci are known to be resistantto bacitracin, and this property was used to isolate mutans streptococcifrom the oral microflora. The MSA agar plates with 0.2 units ofbacitracin per mL were used to determine mutans streptococci.

One-way analysis of variance (ANOVA) was performed to detect thesignificant effects of the variables. Tukey's multiple comparison wasused to compare the data at a p value of 0.05.

Results

FIG. 4A shows schematic of the bonding procedures, FIG. 4B schematic ofthe bond test, and FIG. 9 human dentin shear bond strength data(mean±sd; n=10). Adding 0.1% NAg into primer and adhesive yielded a bondstrength of (30.7±8.3) MPa, similar to (30.2±5.0) MPa for the control(p>0.1). Further adding 10% NACP into the adhesive slightly increasedthe bond strength to (34.3±7.7) MPa (p>0.1). While 40% NACP slightlydecreased the bond strength, all the six groups had bond strengths thatwere not significantly different (p>0.1).

Typical SEM images of the dentin-adhesive interfaces are shown in FIG.10 for (A) SBMP control, (B) P&A+NAg, A+20NACP, and (C) P&A+NAg,A+40NACP. Numerous resin tags “T” from well-filled dentinal tubules werevisible in all the samples. The resin tags were slightly shorter at 40%NACP than the other groups. “HL” refers to the hybrid layer between theadhesive and the underlying mineralized dentin. At a highermagnification, the NACP nanoparticles were visible in (D) with 20% NACP.Arrows in (D) indicate examples of NACP nanoparticles infiltrated intothe dentinal tubules. This feature became more visible at highermagnifications in (E) and (F), where arrows indicate NACP, whichinfiltrated into not only the straight and smooth tubules (E), but alsothe bent and irregularly-shaped tubules (F).

The MTT metabolic results are plotted in FIG. 11. Control disks hadbiofilms with a high metabolic activity. Incorporation of 0.1% of NAgdecreased the metabolic activity by more than half (p<0.05). Adding NACPto the adhesive did not further significantly decrease the metabolicactivity, although there was a decreasing trend at 30% and 40% NACP(p>0.1).

FIG. 12 plots biofilm CFU counts per disk for: (A) Total microorganisms,(B) total streptococci, and (C) mutans streptococci. NAg greatly reducedthe CFU compared to that of the control (p<0.05). Specimens with NAg andNACP reduced the CFU by an order of magnitude, compared to the control.Specimens with 40% NACP slightly reduced the CFU, compared to P&A+NAgwithout NACP; however, this decrease was not statistically significant(p>0.1).

FIG. 13 plots the lactic acid production by biofilms. Biofilms oncontrol disks produced the most acid, indicating that the un-modifiedcommercial bonding agent was not antibacterial. Incorporation of NAgdramatically decreased the acid production, to less than half of that ofthe control (p<0.05). Adding 30% and 40% NACP slightly and significantly(p<0.05) decreased the acid production, compared to P&A+NAg withoutNACP. Lactic acid production by biofilms on the disks with 30% and 40%NACP were about ¼ of the acid production for the control.

Example 4: Dentin Shear Bond Strength of Anti-Bacterial, Re-MineralizingPrimer/Adhesive Dental Bonding Agents

Dental primer/adhesive bonding agents were prepared as described below,and dentin shear bond strength was measured as described in thepreceding Examples. The results are provided in FIG. 14.

Group 1: Scotchbond Multi-Purpose (3M, St. Paul, Minn.), referred to as“SBMP control”.

Group 2: Experimental Primer=PMGDM/HEMA at 3.3/1 ratio+1% BAPO+50%acetone (Referred to as “P control”). Experimental Adhesive=BisGMA/TEGMAat 7/3 ratio+1% BAPO (Referred to as “A control”).

Group 3: P control+0.1% nano silver. A control was not modified.Referred to as “P+NAg, A control”.

Group 4: P control+0.1% nano silver. A control+0.1% nano silver+10%QADM. Referred to as “P+NAg, A+NAg+QADM”.

Group 5: P control+0.1% nano silver. A control+0.1% nano silver+10%QADM+10% NACP. Referred to as “P+NAg, A+NAg+QADM+10NACP”.

Group 6: P control+0.1% nano silver. A control+0.1% nano silver+20%QADM+20% NACP. Referred to as “P+NAg, A+NAg+QADM+20NACP”.

Group 7: P control+0.1% nano silver. A control+0.1% nano silver+10%QADM+30% NACP. Referred to as “P+NAg, A+NAg+QADM+30NACP”.

Group 8: P control+0.1% nano silver. A control+0.1% nano silver+10%QADM+40% NACP. Referred to as “P+NAg, A+NAg+QADM+40NACP”.

Each value is mean±sd; n=10). Horizontal lines in FIG. 14 indicatevalues that are not significantly different (p>0.1). These resultsdemonstrate that the antibacterial and remineralizing bonding agents ofthe invention have the promise of achieving antibacterial andremineralizing capabilities in an experimental bonding agent withoutcompromising the dentin bond strength.

Example 5: Synthesis of Quaternary Ammonium Methacrylates with DifferentAlkyl Chain Lengths

Synthesis of a variety of quaternary ammonium salt (QAS)monomethacrylates was carried out using a Menschutkin reaction(Menschutkin et al. 1890; Antonucci et al. 2012). The reaction proceedsby the addition reaction of tertiary amines with organo-halides. To forma QAS with a reactive methacrylate groups, 2-(dimethylamino)ethylmethacrylate (DMAEMA) was chosen as methacrylate-containing tertiaryamine. In order to investigate the effect of chain length from thequaternary ammonium site on the antibacterial properties of QASmonomers, seven different alkyl organo-halides were each chosen to reactwith BEMA.

Examples are summarized in Table 2. Other chain lengths including 2, 4,5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 23, 24, and 25 can besimilarly synthesized.

TABLE 2 Summary of the Reaction of Dimethylamino methacrylate (DMAEMA)with Various Organo-Halides Alkyl Chain Tertiary Amine AlkylOrgano-Halide Product Length Dimethylamino 1-bromopropane (BP) DMAPM 3methacrylate 1-bromohexane (BH) DMAHM 6 (DMAEMA) 1-bromoheptane (BHP)DMAHPM 7 1-bromooctane (BO) DMAOM 8 1-bromononane (BN) DMANM 91-bromodecane (BD) DMADM 10 1-bromoundecane (BUD) DMAUDM 111-bromododecane (BDD) DMADDM 12 1-bromotridecane (BTD) DMATDM 131-bromotetradecane (BTTD) DMATTDM 14 1-bromopentadecane (BPD) DMAPDM 151-bromohexadecane (BHD) DMAHDM 16 1-bromoheptadecane (BHPD) DMAHPDM 171-bromooctadecane (BOD) DMAODM 18 1-bromononadecane (BND) DMANDM 191-bromoicosane (BIO) DMAIOM 20 1-bromohenicosane (BHO) DMAHOM 211-bromodocosane (BDO) DMADOM 22Synthesis of QAS with Chain Length=3

In a 20 mL scintillation vial, 10 mmol of 2-(dimethylamino)ethylmethacrylate (DMAEMA, Sigma Aldrich, St. Louis Mo.) and 10 mmol of1-bromopropane (BP, TCI America, Portland Oreg.) were added. To thismixture, 3 g of ethanol was added as a solvent. A magnetic stir bar wasadded, and the vial was capped and stirred at 70° C. for 24 h. After thereaction was complete, the ethanol solvent was removed via evaporationat room temperature over several days.

Synthesis of QAS with Chain Length=6

In a 20 mL scintillation vial, 10 mmol of DMAEMA and 10 mmol of1-bromohexane (BH, TCI America, Portland Oreg.) were added. To thismixture, 3 g of ethanol was added as a solvent. A magnetic stir bar wasadded, and the vial was capped and stirred at 70° C. for 24 h. After thereaction was complete, the ethanol solvent was removed via evaporationat room temperature over several days.

Synthesis of QAS with Chain Length=9

In a 20 mL scintillation vial, 10 mmol of DMAEMA and 10 mmol of1-bromononane (BN, TCI America, Portland Oreg.) were added. To thismixture, 3 g of ethanol was added as a solvent. A magnetic stir bar wasadded, and the vial was capped and stirred at 70° C. for 24 h. After thereaction was complete, the ethanol solvent was removed via evaporationat room temperature over several days.

Synthesis of QAS with Chain Length=12

In a 20 mL scintillation vial, 10 mmol of DMAEMA and 10 mmol of1-bromododecane (BDD, TCI America, Portland Oreg.) were added. To thismixture, 3 g of ethanol was added as a solvent. A magnetic stir bar wasadded, and the vial was capped and stirred at 70° C. for 24 h. After thereaction was complete, the ethanol solvent was removed via evaporationat room temperature over several days.

Synthesis of QAS with Chain Length=15

In a 20 mL scintillation vial, 10 mmol of DMAEMA and 10 mmol of1-bromopentadecane (BPD, TCI America, Portland Oreg.) were added. Tothis mixture, 3 g of ethanol was added as a solvent. A magnetic stir barwas added, and the vial was capped and stirred at 70° C. for 24 h. Afterthe reaction was complete, the ethanol solvent was removed viaevaporation at room temperature over several days.

Synthesis of QAS with Chain Length=18

In a 20 mL scintillation vial, 10 mmol of DMAEMA and 10 mmol of1-bromooctadecane (BOD, TCI America, Portland Oreg.) were added. To thismixture, 3 g of ethanol was added as a solvent. A magnetic stir bar wasadded, and the vial was capped and stirred at 70° C. for 24 h. After thereaction was complete, the ethanol solvent was removed via evaporationat room temperature over several days.

Synthesis of QAS with Chain Length=22

In a 20 mL scintillation vial, 10 mmol of DMAEMA and 10 mmol of1-bromodocosane (BDO, TCI America, Portland Oreg.) were added. To thismixture, 3 g of ethanol was added as a solvent. A magnetic stir bar wasadded, and the vial was capped and stirred at 70° C. for 24 h. After thereaction was complete, the ethanol solvent was removed via evaporationat room temperature over several days.

Characterization of Reaction Products

FTIR spectra (Nicolet 6700, Thermo Scientific, Waltham, Mass.) of thestarting materials and the viscous products were collected between twoKBr windows in the 4000 cm⁻¹ to 400 cm⁻¹ region with 128 scans at 4 cm⁻¹resolution. Water and CO₂ bands were removed from all spectra bysubtraction. ¹H NMR spectra (GSX 270, JEOL USA Inc., Peabody, Mass.) ofthe starting materials and products were taken in deuterated chloroformat a concentration of approximately 3%. All spectra were run at roomtemperature, 15 Hz sample spinning, 45° tip angle for the observationpulse, and a 10 s recycle delay, for 64 scans.

Cytotoxicity of Monomers Via MTT Assay

Human gingival fibroblasts (HGF, ScienCell) were cultured in afibroblast medium (FM). Each unpolymerized monomer was dissolved in FM,at concentrations of: 0 (control), 0.5, 1, 2, 5, 10, 20, 40, 60, and 100μg/mL (Huang L et al. 2011; Chai Z et al. 2011). Then, HGF were seededin 96-well plates at 5,000 cells per well. After 2 d, 20 μL of MTTsolution was added (Chai Z et al. 2011). After 4 h, the unreacted dyewas removed and 150 μL of dimethyl sulfoxide was added. Absorbance wasmeasured via the microplate reader at 492 nm. Relative fibroblastviability=absorbance of monomer sample/absorbance of control withoutmonomer (Chai Z et al. 2011). The results are provided in FIG. 15A.Cells were also live/dead stained (Molecular Probes) and examined withfluorescence microscopy (TE2000-S, Nikon) as shown in FIG. 15B.

Incorporating Antibacterial Monomers with Different Chain Lengths intoDental Composites

The composite matrix is a resin or combinations of resins selected fromthe group consisting of bis-GMA (bisphenol glycidyl methacrylate),TEGDMA (triethylene glycol dimethacrylate), HEMA (2-hydroxyethylmethacrylate), UDMA (urethane dimethacrylate) and PMGDM (pyromelliticacid glycerol dimethacrylate). The composite fillers may include calciumphosphate nanoparticles such as nanoparticles of amorphous calciumphosphate (NACP). The NACP particles range in size from about 10 nm toabout 500 nm. The NACP filler level ranges from about 5% to about 90% ofthe mass of the composite. The composite can contain other fillers suchas usual dental glass fillers. Alternatively, the composite may containglass fillers, without calcium phosphate fillers, in which theincorporation of the new antibacterial monomers will render thecomposite strongly antibacterial. The new antibacterial composite maycontain fibers and whiskers as mechanical reinforcement.

One or more antibacterial monomers with various chain lengths can beincorporated into the composite, at antibacterial resin mass fractionsranging from 1% to 50% of the composite, preferably 2% to 20% of thecomposite. Other techniques for producing the dental composites aredisclosed in WO 2012/003290, incorporated herein by reference in itsentirety.

Data on Longer Chain Length

A new antibacterial monomer with a chain length of 16 was also preparedwhich exhibited a MBC=0.61 μg/mL and a MIC=0.305 μg/mL. These values arean order of magnitude more potent than those for chain length 12reported below.

Example 6: Alternative Means for Synthesis of Quaternary AmmoniumMethacrylates (QAMs)

A modified Menschutkin reaction approach was used to synthesize the newQAMs. This method uses a tertiary amine group to react with anorgano-halide, as described in previous studies (Antonucci J M et al.2012; Cheng L et al. 2012a). A benefit of this reaction is that thereaction products are generated at virtually quantitative amounts andrequire minimal purification (Antonucci J M et al. 2012). In the presentstudy, 2-bromoethyl methacrylate (BEMA) was the organo halide.N,N-dimethylaminohexane (DMAH) and 1-(dimethylamino) docecane (DMAD)were the two tertiary amines.

The scheme of synthesis of dimethylaminohexane methacrylate (DMAHM) isshown in FIG. 16A. Ten mmol of DMAH (Tokyo Chemical Industry, Tokyo,Japan), 10 mmol of BEMA (Monomer-Polymer and Dajac Labs, Trevose, Pa.),and 3 g of ethanol were added to a 20 mL scintillation vial with amagnetic stir bar. The vial was capped and stirred at 70° C. for 24 h.After the reaction was complete, the ethanol solvent was removed viaevaporation at room temperature over several days. This yielded DMAHM asa clear liquid.

The scheme of synthesis of dimethylaminododecyl methacrylate (DMADDM) isshown in FIG. 16B. In a 20 mL scintillation vials were added 10 mmol ofDMAD (Tokyo Chemical Industry), 10 mmol of BEMA, and 3 g of ethanol. Amagnetic stir bar was added, and the vial was capped and stirred at 70°C. for 24 h. After the reaction was complete, the solvent was removedvia evaporation. The number of the alkyl chain length units was 6 forDMAHM and 12 for DMADDM (FIG. 16).

To characterize the reaction products, Fourier transform infraredspectroscopy (FTIR, Nicolet 6700, Thermo Scientific, Waltham, Mass.) wasused. FTIR spectra of the starting materials and the viscous productswere collected between two KBr windows in the 4000 cm⁻¹ to 400 cm⁻¹region with 128 scans at 4 cm⁻¹ resolution (Antonucci J M et al. 2012).Water and CO₂ bands were removed from all spectra by subtraction. ¹H NMRspectra (GSX 270, JEOL, Peabody, Mass.) of the starting materials andproducts were taken in deuterated chloroform at a concentration ofapproximately 3%. All spectra were run at room temperature, 15 Hz samplespinning, 45° tip angle for the observation pulse, and a 10 s recycledelay, for 64 scans (Antonucci J M et al. 2012).

Representative results are provided in FIG. 17. The characterizationusing FTIR and ¹H NMR indicated that the Menschutkin reaction wassuccessful. The infrared spectroscopy showed the disappearance of C—Brand tertiary amine groups, and the appearance of quaternary ammoniumgroup that resulted from the reaction. In each plot, the appearance ofthe NR₄ ⁺ peak in the last curve corresponded to the formation of thequaternary ammonium group. In FIG. 17A, FTIR showed that the C—Brabsorption bands from BEMA (575 cm⁻¹, 512 cm⁻¹) in curve 1 and the(CH₃)₂N⁻ bands (2822 cm⁻¹, 2771 cm⁻¹) from DMAH in curve 2 disappearedin curve 3. This indicated that the bromine group in BEMA successfullyreacted with the amine group in DMHA to form the quaternary ammoniumgroup. The appearance in curve 3 of the NR₄ ⁺ peak corresponded to theformation of the quaternary ammonium group and, hence, DMAHM wassuccessfully synthesized. Similarly, FIG. 17B showed the synthesis ofDMADDM from the reaction of BEMA and DMAD.

Minimum Inhibitory Concentration (MIC) and Bactericidal Concentration(MBC)

MIC and MBC were measured using S. mutans (ATCC 700610, UA159, AmericanType Culture, Manassas, Va.). S. mutans is a cariogenic, aerotolerantanaerobic bacterium and the primary causative agent of dental caries(Loesche 1986). MIC and MBC were determined via serial microdilutionassays (Imazato S et al. 2006; Huang L et al. 2011). Unpolymerized DMAHMor DMADM monomer was dissolved in brain heart infusion (BHI) broth (BD,Franklin Lakes, N.J.) to give a final concentration of 200 mg/mL. Fromthese starting solutions, serial two fold dilutions were made into 1 mLvolumes of BHI broth. 15 μL of stock S. mutans was added to 15 mL of BHIbroth with 0.2% sucrose and incubated at 37° C. with 5% CO₂. Overnightcultures of S. mutans were adjusted to 2×10⁶ CFU/mL with BHI broth, and50 μL of inocula was added to each well of a 96-well plate containing 50μL of a series of antibacterial monomer dilution broths. BHI broth with1×10⁶ CFU/mL bacteria suspension without antibacterial agent served asnegative control. Chlorhexidine diacetate (CHX) (Sigma, St. Louis, Mo.)served as positive control. The previously-synthesized QADM (Antonucci JM et al. 2012; Cheng L et al. 2012a) served as an antibacterial monomercontrol. After incubation at 37° C. in 5% CO₂ for 48 h, the wells wereread for turbidity, referenced by the negative and positive controlwells. MIC was determined as the endpoint (the well with the lowestantibacterial agent concentration) where no turbidity could be detectedwith respect to the controls (Huang L et al. 2011). To determine MBC, analiquot of 50 μL from each well without turbidity was inoculated on BHIagar plates and incubated at 37° C. in 5% CO₂ for 48 h. MBC wasdetermined as the lowest concentration of antibacterial agent thatproduced no colonies on the plate. The tests were performed intriplicate (Huang L et al. 2011). The MIC and MBC values of theantibacterial agents against S. mutans are listed in Table 3.

TABLE 3 MIC and MBC values of various antibacterial agents against S.mutans* Compound MBC MIC QADM 2.5 × 10⁴ μg/mL 1.25 × 10⁴ μg/mL DMAHM3.13 × 10³ μg/mL 1.56 × 10³ μg/mL DMADDM 12.21 μg/mL 6.10 μg/mL CHX 3.91μg/mL 1.95 μg/mL *CHX = Chlorhexidine. QADM = quaternary ammoniumdimethacrylate. DMAHM = dimethylaminohexane methacrylate. DMADDM =dimethylaminododecyl methacrylate. Tests were repeated in triplicate.

A lower concentration of the antibacterial agent needed to inhibit thebacteria indicates a higher antibacterial potency. The new DMAHM with analkyl chain length of 6 was more potent than the previously-synthesizedQADM. In dramatic contrast, the new DMADDM with an alkyl chain length of12 was much more strongly antibacterial than DMAHM. The MIC and MBC ofDMADDM was more than two orders of magnitude lower than those of MDAHM,and approached those of the CHX control.

Processing of DMADDM-NACP Nanocomposite

A spray-drying technique as described previously in Chow L C et al.(2004) was used to make NACP (Ca₃[PO₄]₂). Calcium carbonate (CaCO₃,Fisher, Fair Lawn, N.J.) and dicalcium phosphate anhydrous (CaHPO₄,Baker Chemical, Phillipsburg, N.J.) were dissolved into an acetic acidsolution to obtain final Ca and P ionic concentrations of 8 mmol/L and5.333 mmol/L, respectively. This resulted in a Ca/P molar ratio of 1.5,the same as that for ACP. This solution was sprayed into a heatedchamber, and an electrostatic precipitator (AirQuality, Minneapolis,Minn.) was used to collect the dried particles. This method producedNACP with a mean particle size of 116 nm, as measured in a previousstudy (Xu H H K et al. 2011). Other techniques for producing NACP aredisclosed in WO 2012/003290, incorporated herein by reference in itsentirety.

Because DMADDM exhibited a much greater antibacterial potency than DMAHMand QADM, DMADDM was used for incorporation into the NACP nanocompositeto obtain antibacterial properties. BisGMA (bisphenol glycidyldimethacrylate) and TEGDMA (triethylene glycol dimethacrylate) (Esstech,Essington, Pa.) were mixed at a mass ratio=1:1, and renderedlight-curable with 0.2% camphorquinone and 0.8% ethyl4-N,N-dimethylaminobenzoate (mass fractions). DMADDM was mixed with thephoto-activated BisGMA-TEGDMA resin at the followingDMADDM/(BisGMA-TEGDMA+DMADDM) mass fractions: 0%, 2.5%, 5%, 7.5% and10%, yielding five groups of resin, respectively. A dental bariumboroaluminosilicate glass of a median particle size of 1.4 μm(Caulk/Dentsply, Milford, Del.) was silanized with 4%3-methacryloxypropyltrimethoxysilane and 2% n-propylamine (Xu H H K etal. 2011). The NACP and glass particles were mixed into each resin, atthe same filler level of 70% by mass, with 20% of NACP and 50% of glass(Xu H H K et al. 2011). Because the resin mass fraction was 30% in thecomposite, the five DMADDM mass fractions in the composite were 0%,0.75%, 1.5%, 2.25% and 3%, respectively. Other techniques for producingthe dental composites are disclosed in WO 2012/003290, incorporatedherein by reference in its entirety.

Six composites were tested: Five NACP nanocomposites at the five DMADDMmass fractions described above, and a commercial control composite.Renamel (Cosmedent, Chicago, Ill.) served as a control composite. Itconsisted of nanofillers of 20-40 nm in size, at 60% filler level in amultifunctional methacrylate ester resin. For mechanical testing, eachpaste was placed into rectangular molds of 2×2×25 mm. For biofilmexperiments, each paste was placed into disk molds of 9 mm in diameterand 2 mm in thickness. The specimens were photo-cured (Triad 2000,Dentsply, York, Pa.) for 1 min on each side. The specimens were thenincubated in distilled water at 37° C. for 24 hours prior to mechanicalor biofilm testing.

Mechanical Testing

A computer-controlled Universal Testing Machine (5500R, MTS, Cary, N.C.)was used to fracture the specimens in three-point flexure using a spanof 10 mm and a crosshead speed of 1 mm/min. Flexural strength S wasmeasured as: S=3P_(max)L/(2bh²), where P_(max) is the load-at-failure, Lis span, b is specimen width and h is specimen thickness. Elasticmodulus E was measured as: E=(P/d)(L³/[4bh³]), where load P divided bydisplacement d is the slope in the linear elastic region of theload-displacement curve. The specimens were taken out of the water andfractured within several minutes while still being wet (Cheng L et al.2012a).

FIG. 18 plots (A) flexural strength, and (B) elastic modulus of thecomposites (mean±sd; n=6). The NACP nanocomposite with various DMADDMmass fractions had strengths similar to that of the commercial compositecontrol, which was not antibacterial and had no Ca and P ion release(p>0.1). The elastic moduli of DMADDM-NACP nanocomposites were alsosimilar to those of the NACP nanocomposite without DMADDM and thecomposite control (p>0.1).

Dental Plaque Microcosm Biofilm and Live/Dead Assay

The dental plaque microcosm biofilm model used human saliva as inoculum.Saliva was collected from a healthy adult donor following a previousstudy (Cheng L et al. 2012b). The donor had natural dentition withoutactive caries or periopathology, and without the use of antibioticswithin the last 3 months. The donor did not brush teeth for 24 h andabstained from food or drink intake for at least 2 h prior to donatingsaliva (Cheng L et al. 2012b). Stimulated saliva was collected duringparafilm chewing and kept on ice. The saliva was diluted in sterileglycerol to a concentration of 70% saliva and 30% glycerol (Cheng L etal. 2012b), and stored at −80° C.

The saliva-glycerol stock was added, with 1:50 final dilution, into thegrowth medium as inoculum. The growth medium contained mucin (type II,porcine, gastric) at a concentration of 2.5 g/L; bacteriologicalpeptone, 2.0 g/L; tryptone, 2.0 g/L; yeast extract, 1.0 g/L; NaCl, 0.35g/L, KCl, 0.2 g/L; CaCl₂, 0.2 g/L; cysteine hydrochloride, 0.1 g/L;haemin, 0.001 g/L; vitamin K₁, 0.0002 g/L, at pH 7 (McBain A J 2009).Composite disks were sterilized in ethylene oxide (Anprolene AN 74i,Andersen, Haw River, N.C.). 1.5 mL of inoculum was added to each well of24-well plates with a composite disk, and incubated in 5% CO₂ at 37° C.for 8 h. The disks were then transferred to new 24-well plates filledwith fresh medium and incubated. After 16 h, the disks were transferredto new 24-well plates with fresh medium and incubated for 24 h. Thistotaled 48 h of incubation, which was shown to be adequate to formdental plaque microcosm biofilms on resins (Cheng L et al. 2012b; ZhangK et al. 2012).

After 48 h of growth, the microcosm biofilms adherent on the disks weregently washed three times with phosphate buffered saline (PBS), and thenstained using the BacLight live/dead bacterial viability kit (MolecularProbes, Eugene, Oreg.) (Cheng L et al. 2012b; Zhang K et al. 2012). Livebacteria were stained with Syto 9 to produce a green fluorescence, andbacteria with compromised membranes were stained with propidium iodideto produce a red fluorescence. The stained disks were examined using aconfocal laser scanning microscopy (CLSM 510, Carl Zeiss, Thornwood,N.Y.).

Biofilms on composite control and NACP nanocomposite without DMADDM hadprimarily live bacteria (data not shown). Increasing the DMADDM massfraction in the nanocomposite resulted in much more red/yellow/orangestaining, indicating that the DMADDM-containing nanocompositeseffectively inhibited the biofilm growth. These results also indicatethat NACP was not antibacterial, and DMADDM was responsible for theantibacterial activity.

MTT Assays

MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assaywas performed according to previous studies (Antonucci J M et al. 2012;Cheng L et al. 2012a). It is a colorimetric method that measures theenzymatic reduction of MTT, a yellow tetrazole, to formazan. Briefly,disks with 48-h biofilms were rinsed with PBS and transferred to 24 wellplates. Then, 1 mL of MTT dye (0.5 mg/mL MTT in PBS) was added to eachwell and incubated for 1 h. The disks were transferred to new 24-wellplates, 1 mL of dimethyl sulfoxide (DMSO) was added to solubilize theformazan crystals, and the plate was incubated for 20 min in the dark.Then, 200 μL of the DMSO solution from each well was transferred to a96-well plate, and the absorbance at 540 nm was measured via amicroplate reader (SpectraMax M5, Molecular Devices, Sunnvale, Calif.)(Cheng L et al. 2012a).

FIG. 19 plots (A) the MTT assay, and (B) lactic acid production ofbiofilms adherent on the composites. Each values is mean±sd (n=6). In(A), the biofilms on composite control and NACP+0% DMADDM had a similarmetabolic activity (p>0.1). Increasing the DMADDM mass fractionsignificantly decreased the metabolic activity of biofilms (p<0.05). At3% DMADDM in the composite, the metabolic activity was approximately 5%of that on composite control. In (B), the biofilms on composite controlproduced the most acid, similar to that on NACP+0% DMADDM. Withincreasing DMADDM mass fraction, the lactic acid productionmonotonically decreased (p<0.05). The lactic acid production by biofilmson NACP+3% DMADDM was about 1% of that on the commercial compositecontrol.

Lactic Acid Production and CFU Counts

Composite disks with 48-h biofilms were rinsed in cysteine peptone water(CPW) to remove the loose bacteria. Each disk was placed in a new24-well plate and 1.5 mL of buffered peptone water (BPW) supplementedwith 0.2% sucrose (Cheng L et al. 2012a). The samples were incubated in5% CO₂ at 37° C. for 3 h to allow the biofilms to produce acid. The BPWsolutions were then stored for lactate analysis. Lactate concentrationswere determined using an enzymatic (lactate dehydrogenase) methodaccording to previous studies (Cheng L et al. 2012a; Cheng L et al.2012b). The microplate reader was used to measure the absorbance at 340nm for the collected BPW solutions. Standard curves were prepared usinga lactic acid standard (Supelco Analytical, Bellefonte, Pa.) (Cheng L etal. 2012a; Cheng L et al. 2012b).

Composite disks with 2-day biofilms were transferred into tubes with 2mL CPW, and the biofilms were harvested by sonication and vortexing atthe maximum speed for 20 seconds using a vortex mixer (Fisher,Pittsburgh, Pa.). Three types of agar plates were used to assess themicroorganism viability after serial dilution in CPW: Mitis salivariusagar (MSA) culture plates, containing 15% sucrose, to determine totalstreptococci (Lima J P et al. 2009); MSA agar culture plates plus 0.2units of bacitracin per mL, to determine mutans streptococci (Park J Het al. 2006); and Tryptic Soy Blood Agar culture plates to determinetotal microorganisms (Cheng L et al. 2012b). One-way analysis ofvariance (ANOVA) was performed to detect the significant effects of thevariables. Tukey's multiple comparison test was used at a p value of0.05.

FIG. 20 plots the CFU counts for: (A) Total microorganisms, (B) totalstreptococci, and (C) mutans streptococci (mean±sd; n=6). The compositecontrol had the highest CFU counts. All three CFU counts showed asimilar decreasing trend with increasing DMADM mass fraction in NACPnanocomposite (p<0.05). Compared to the control composite, all three CFUcounts on NACP+3% DMADDM were reduced by 2-3 orders of magnitude.

The present study demonstrated that the antibacterial monomers, such asDMADDM, could be incorporated into the NACP nanocomposite to impart astrong antibacterial activity without compromising mechanicalproperties. This indicates the versatility of incorporating varioustypes of antibacterial monomers into the NACP nanocomposite, and themiscibility and compatibility of the antibacterial monomers with NACPnanocomposite. It is interesting to compare the DMADDM nanocomposite ofthe present study with the previous QADM nanocomposite tested by thesame operator using the same procedures (Cheng et al. 2012c). Theprevious QADM nanocomposite reduced the MTT metabolic activity by2-fold, compared to the same control composite (Cheng L et al. 2012c).The present study using DMADDM reduced the MTT by 20-fold. In addition,the previous QADM nanocomposite reduced the lactic acid production by2-fold (Cheng L et al. 2012c); the present study using DMADDM reducedlactic acid by 2 orders of magnitude. Furthermore, the previous QADMnanocomposite reduced the biofilm CFU counts by 3-fold (Cheng L et al.2012c); the present study using DMADDM reduced the biofilm CFU by 2-3orders of magnitude. Therefore, the new DMADDM-NACP nanocompositerepresents a substantial improvement over previous antibacterial dentalcomposites.

Example 7: DMADDM Bond Strength Durability at 1 Day and at 6 Months

Extracted human molar teeth were used (n=10). The monomer DMADDM andnanoparticles of silver (NAg) were incorporated into a commercialbonding agent SBMP. The bonded dentin samples were immersed in water for1 day and 6 months, to test the degradation of bond strength over time.The commercial control lost ⅓ of its strength in 6 months, which isunusual for commercial bonding agents (FIG. 21). In contrast, theantibacterial bonding agents showed no loss of dentin bond strength.These results demonstrate the double benefits of the new antibacterialbonding agents: inhibiting biofilms and caries, and improving thedurability of the mechanical strength of the bonded interface.

Example 8

The objectives of the study were to develop antibacterial dental primerand dental adhesive containing a new quaternary ammonium monomer(dimethylaminododecyl methacrylate, DMADDM) as well as nanoparticles ofsilver (NAg), and to investigate their effects on antibacterial anddentin bond properties. The minimum inhibitory concentration (MIC) andminimum bactericidal concentration (MBC) of the new DMADDM were ordersof magnitude lower than those of a previously-synthesized quaternaryammonium dimethacrylates (QADM). Un-cured dental primer containingDMADDM produced much larger bacteria inhibition zones than QADM(p<0.05). Cured primer/adhesive samples containing DMADDM and NAggreatly reduced biofilm metabolic activity (p<0.05). Combining DMADDMwith NAg in primer/adhesive resulted in less CFU than DMADDM alone(p<0.05). Lactic acid production by biofilms was reduced by 20-fold viaDMADDM and NAg, compared to commercial bonding agent control.Incorporation of the new DMADDM and NAg into primer and adhesive did notadversely affect the dentin bond strength.

Materials and Methods Developing New Antibacterial Monomers

Two new antibacterial monomers were synthesized: dimethylaminohexanemethacrylate (DMAHM) with an alkyl chain length of 6, anddimethylaminododecyl methacrylate (DMADDM) with an alkyl chain length of12. A modified Menschutkin reaction method was employed, which used atertiary amine group to react with an organo-halide, following previousstudies (Antonucci et al. 2012; Cheng et al. 2012a; Cheng et al. 2012a).This method is useful because the reaction products are generated atvirtually quantitative amounts and require minimal purification(Antonucci et al. 2012). To synthesize DMAHM, 2-bromoethyl methacrylate(BEMA) served as the organo halide, and N,N-dimethylaminohexane (DMAH)served as the tertiary amine. Ten mmol of DMAH (Tokyo Chemical Industry,Tokyo, Japan), 10 mmol of BEMA (Monomer-Polymer and Dajac Labs, Trevose,Pa.), and 3 g of ethanol were added to a 20 mL scintillation vial with amagnetic stir bar. The vial was capped and stirred at 70° C. for 24 h.After the reaction was complete, the ethanol solvent was removed viaevaporation, yielding DMAHM as a clear, colorless, and viscous liquid.To synthesize the second new monomer DMADDM, BEMA was the organo halide,and 1-(dimethylamino)docecane (DMAD) was the tertiary amine. In a 20 mLscintillation vials were added 10 mmol of DMAD (Tokyo Chemical Industry)and 10 mmol of BEMA, while otherwise following the same procedures asfor DMAHM. Fourier transform infrared (FTIR) spectroscopy (Nicolet 6700,Thermo Scientific, Waltham, Mass.) spectra of the starting materials andthe products were collected between two KBr windows in the 4000 to 400cm⁻¹ region. ¹H NMR spectra (GSX 270, JEOL) were taken in deuteratedchloroform at a concentration of about 3% (Antonucci et al. 2012). Thereactions and products of DMAHM and DMADDM were all verified inpreliminary studies.

Minimum Inhibitory Concentration (MIC) and Minimum BactericidalConcentration (MBC)

The use of S. mutans (ATCC 700610, American Type Culture, Manassas, Va.)was approved by the University of Maryland. S. mutans is a cariogenic,aerotolerant anaerobic bacterium and the primary causative agent ofdental caries (Deng et al. 2004). MIC and MBC were determined via serialmicrodilution assays (Imazato et al. 2006; Huang et al. 2011).Unpolymerized DMAHM or DMADDM monomer was dissolved in brain heartinfusion (BHI) broth (BD, Franklin Lakes, N.J.) to a concentration of200 mg/mL. From these starting solutions, serial two fold dilutions weremade into 1 mL volumes of BHI broth. Fifteen μL of stock S. mutans wasadded to 15 mL of BHI broth with 0.2% sucrose and incubated at 37° C.with 5% CO₂. Overnight cultures of S. mutans were adjusted to 2×10⁶CFU/mL with BHI, and 50 μL of inoculum was added to each well of a96-well plate containing 50 μL of a series of antibacterial monomerdilution broths. BHI with 1×10⁶ CFU/mL bacteria suspension withoutantibacterial agent served as negative control. Chlorhexidine diacetate(CHX) (Sigma, St. Louis, Mo.) served as positive control. Thepreviously-synthesized QADM (Antonucci et al. 2012; Cheng et al. 2012a)served as an antibacterial monomer control. The wells were read forturbidity after incubation at 37° C. in 5% CO₂ for 48 h, referenced bythe negative and positive control wells. MIC was defined as the endpoint(the well with the lowest antibacterial agent concentration) where noturbidity could be detected with respect to the controls (Huang et al.2011). An aliquot of 50 μL from each well without turbidity wasinoculated on BHI agar plates. After incubation at 37° C. in 5% CO₂ for48 h, the MBC value was defined as the lowest concentration ofantibacterial agent that produced no colonies on the plate. The testswere performed in triplicate (Huang et al. 2011).

Fabrication of Antibacterial Primer and Adhesive

Scotchbond Multi-Purpose (3M, St. Paul, Minn.), referred to as “SBMP”,was used as the parent bonding system to test the effect ofincorporation of antibacterial agents. According to the manufacturer,SBMP etchant contains 37% phosphoric acid. SBMP primer contains 35-45%2-Hydroxyethylmethacrylate (HEMA), 10-20% copolymer of acrylic anditaconic acids, and 40-50% water. SBMP adhesive contains 60-70% BisGMAand 30-40% HEMA.

DMAHM was mixed with SBMP primer at a DMAHM/(primer+DMAHM) mass fractionof 5%. The 5% was selected following a previous study (Imazato et al.2006). Similarly, 5% of DMAHM was incorporated into the SBMP adhesive.The second new monomer, DMADDM, was also incorporated into the SBMPprimer and adhesive at 5% mass fraction.

Another antibacterial agent NAg was also incorporated into the primerand adhesive. Silver 2-ethylhexanoate powder (Strem, New Buryport,Mass.) was dissolved in 2-(tert-butylamino)ethyl methacrylate (TBAEMA,Sigma) at 0.1 g of silver salt per 0.9 g of TBAEMA (Cheng et al. 2012a;Cheng Y J. et al. 2011). TBAEMA was used because it improves thesolubility by forming Ag—N coordination bonds with Ag ions, therebyfacilitating the Ag salt to dissolve in the resin solution. TBAEMA wasselected since it contains reactive methacrylate groups and thereforecan be chemically incorporated into a dental resin uponphoto-polymerization (Cheng Y J. et al. 2011). This method produced NAgwith a mean particle size of 2.7 nm that were well dispersed in theresin matrix (Cheng et al. 2012a; Cheng Y J. et al. 2011). The Agsolution was mixed with SBMP primer at a silver2-ethylhexanoate/(primer+silver 2-ethylhexanoate) mass fraction of 0.1%,following a previous study (Zhang et al. 2012). The same 0.1% was usedin the SBMP adhesive to formulate the antibacterial adhesive.

Dental Plaque Microcosm Model

Human saliva is useful for growing plaque microcosm biofilms in vitro tohave the advantage of maintaining much of the complexity andheterogeneity of in vivo plaques (McBain. et al. 2009). The dentalplaque microcosm model was approved by the University of Maryland.Saliva was collected from a healthy adult donor having natural dentitionwithout active caries or periopathology, and without the use ofantibiotics within the past three months (Cheng et al. 2012b). The donordid not brush teeth for 24 h and abstained from food/drink intake for atleast 2 h prior to donating saliva. Stimulated saliva was collectedduring parafilm chewing and kept on ice. Saliva was diluted in sterileglycerol to a saliva concentration of 70% and stored at −80° C. (Chenget al. 2012b).

Agar Disk-Diffusion Test of Uncured Antibacterial Primers

Agar disk diffusion test (ADT) was used to examine the antibacterialeffect of un-cured primers. Five primers were tested: SBMP controlprimer (referred to as “P”); P+5% QADM; P+5% DMAHM; P+5% DMADDM; P+5%DMADDM+0.1% NAg.

Three types of culture media were used: (A) Tryptic Soy Blood Agarculture plates to determine total microorganisms; (B) mitis salivariusagar (MSA) culture plates, containing 15% sucrose, to determine totalstreptococci; (C) MSA agar culture plates plus 0.2 units of bacitracinper mL to determine mutans streptococci. The saliva-glycerol stock wasadded to a growth medium containing mucin (at a concentration of 2.5g/L), bacteriological peptone (2.0 g/L), tryptone (2.0 g/L), yeastextract (1.0 g/L), NaCl (0.35 g/L), KCl (0.2 g/L), CaCl₂ (0.2 g/L), andcysteine hydrochloride (0.1 g/L), at pH of 7 (McBain et al. 2009). Theinoculum was incubated at 37° C. in 5% CO₂ for 24 h. After 24 h, 0.4 mLof bacteria suspension was swabbed across an agar plate with a diameterof 90 mm. A sterile paper disk with a diameter of 6 mm and a thicknessof 1.5 mm was impregnated with 20 μL of a primer. The primer-impregnatedpaper disk was placed on an agar plate with bacteria, and incubated in5% CO₂ at 37° C. for 48 h. Bacteria inhibition zone size=(Outer diameterof inhibition zone−paper disk diameter)/2 (Imazato et al. 2006).

Specimen Fabrication and Biofilm Culture

The MIC, MBC and ADT results showed that DMADDM was much more stronglyantibacterial than DMAHM and QADM. Therefore, DMADDM was selected forincorporation into SBMP primer and adhesive for the subsequentexperiments.

Six bonding agents were used in biofilm tests:

[i] SBMP control primer P and adhesive A (referred to as “SBMP P & Acontrol”)[ii] P+5% DMADDM, with unmodified adhesive A (referred to as “P+DMADDM,A control”)[iii] P+5% DMADDM+0.1% NAg, unmodified A (“P+DMADDM+NAg, A control”)[iv] A+5% DMADDM, biofilm was cultured on adhesive without primer(“A+DMADDM, no P”)[v] A+5% DMADDM+0.1% NAg, biofilm on adhesive, no primer (“A+DMADDM+NAg,no P”)[vi] 5% DMADDM and 0.1% NAg were added to both A and P(“A&P+DMADDM+NAg”)

The purpose of i-iii was to investigate the new DMADDM and itscombination with NAg in cured primer on antibacterial properties(schematic in FIG. 24A). The purpose of iv and v was to examine theantibacterial effect of adhesive, with biofilms on adhesive withoutprimer (schematic in FIG. 24B). The purpose of vi was to determine theeffect of both primer and adhesive being antibacterial (FIG. 24A),instead of using antibacterial primer alone (iii), or antibacterialadhesive alone (v).

Layered disk specimens for biofilm experiments were fabricated followingprevious studies (Li et al. 2009; Imazato et al. 1998). A polyethylenedisk mold (inner diameter=9 mm, thickness=2 mm) was situated on a glassslide. For groups i, ii, iii and vi, a primer was first applied into themold to cover the glass slide. After drying with a stream of air, anadhesive was applied and cured for 20 s (Optilux VCL 401, Demetron Kerr,Danbury, Conn.). Then, a composite (TPH, Caulk/Dentsply, Milford, Del.)was placed on the adhesive to fill the disk mold and was light-cured for1 min. For groups iv and v, each adhesive was applied into the mold tocover the glass slide. Then, a composite (TPH) was placed onto theadhesive to fill the disk mold and light-cured for 1 min. The disks wereimmersed in sterile water and agitated for 1 h to remove any uncuredmonomer, following a previous study (Imazato et al. 1998). The diskswere then dried and sterilized with ethylene oxide (Anprolene AN 74i,Andersen, Haw River, N.C.).

The saliva-glycerol stock was added, with 1:50 final dilution, into thegrowth medium as inoculum, as described above. A layered disk was placedin a well of 24-well plates, and 1.5 mL of inoculum was added andincubated in 5% CO₂ at 37° C. for 8 h. The disks were then transferredto new 24-well plates filled with fresh medium and incubated. After 16h, the disks were transferred to new 24-well plates with fresh mediumand incubated for 24 h. Microcosm biofilms were formed on the disks withthis 48 h incubation, as shown previously (Cheng et al. 2012b; Zhang etal. 2012).

MTT Assay of Biofilm Metabolic Activity

A MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)assay was used to examine the metabolic activity of biofilms (Cheng etal. 2012a). MTT is a colorimetric assay that measures the enzymaticreduction of MTT, a yellow tetrazole, to formazan. Disks with 2-daybiofilms were transferred to a new 24-well plate, and 1 mL of MTT dye(0.5 mg/mL MTT in PBS) was added to each well and incubated at 37° C. in5% CO₂ for 1 h. During this process, metabolically active bacteriareduced the MTT to purple formazan. After 1 h, the disks weretransferred to a new 24-well plate, 1 mL of dimethyl sulfoxide (DMSO)was added to solubilize the formazan crystals, and the plate wasincubated for 20 min at room temperature in the dark. After mixing viapipetting, 200 μL of the DMSO solution from each well was transferred toa 96-well plate, and the absorbance at 540 nm was measured via amicroplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, Calif.).A higher absorbance is related to a higher formazan concentration, whichindicates a higher metabolic activity in the biofilm on the disk.

Colony Forming Units (CFU) and Lactic Acid Production

Disk with 2-day microcosm biofilms were rinsed with cysteine peptonewater (CPW) to remove loose bacteria. The disks were then transferred to24-well plates containing buffered peptone water (BPW) plus 0.2%sucrose, and incubated in 5% CO₂ at 37° C. for 3 h to allow the biofilmsto produce acid (Cheng et al. 2012a; Cheng et al. 2012b). Subsequently,the BPW solutions were stored for lactate analysis. The disks withbiofilms were then transferred into tubes with 2 mL CPW, and thebiofilms were harvested by sonication and vortexing at the maximum speedfor 20 seconds using a vortex mixer (Fisher, Pittsburgh, Pa.). Threetypes of agar plates were used to measure the CFU counts to assess themicroorganism viability (Cheng et al. 2012b). First, tryptic soy bloodagar culture plates were used to determine total microorganisms. Second,(MSA) culture plates containing 15% sucrose were used to determine totalstreptococci (Lima et al. 2009). Third, MSA agar culture plates plus 0.2units of bacitracin per mL was used to determine mutans streptococci.

The lactate concentrations in the BPW solutions were determined using anenzymatic (lactate dehydrogenase) method (Cheng et al. 2012a; Cheng etal. 2012b). A microplate reader (SpectraMax M5) was used to measure theabsorbance at 340 nm (optical density OD₃₄₀) for the collected BPWsolutions. Standard curves were prepared using a lactic acid standard(Supelco, Bellefonte, Pa.) (Cheng et al. 2012a; Cheng et al. 2012b).

Dentin Shear Bond Strength

The use of extracted human teeth was approved by the University ofMaryland. Caries-free molars were cleaned and stored in 0.01% thymolsolution. Flat mid-coronal dentin surfaces were prepared by cutting offthe tips of crowns with a diamond saw (Isomet, Buehler, Lake Bluff,Ill.). Each tooth was embedded in a poly-carbonate holder (Bosworth,Skokie, Ill.) and ground perpendicular to the longitudinal axis on320-grit silicon carbide paper until the occlusal enamel was removed.The dentin surface was etched with 37% phosphoric acid gel for 15 s andrinsed with water for 15 s (Antonucci et al. 2009). A primer was appliedwith a brush-tipped applicator and rubbed in for 15 s. The solvent wasremoved with a stream of air for 5 s. An adhesive was applied andlight-cured for 10 s (Optilux). A stainless-steel iris, having a centralopening with a diameter of 4 mm and a thickness of 1.5 mm, was heldagainst the adhesive-treated dentin surface. The opening was filled witha composite (TPH) and light-cured for 60 s (Antonucci et al. 2009). Thebonded specimens were stored in water at 37° C. for 24 h. A chiselconnected with a Universal Testing Machine (MTS, Eden Prairie, Minn.)was aligned to be parallel to the composite-dentin interface (Cheng etal. 2012b; Antonucci et al. 2009). The load was applied at a rate of 0.5mm/min until the bond failed. Dentin shear bond strength, S_(D), wascalculated as: S_(D)=4P/(πd²), where P is the load at failure, and d isthe diameter of the composite. Ten teeth were tested for each group(n=10) (Cheng et al. 2012b; Antonucci et al. 2009).

Statistical Analyses

One-way analysis of variance (ANOVA) was performed to detect thesignificant effects of the variables. Tukey's multiple comparison testwas used to compare the data at a p value of 0.05.

Results

The MIC and MBC results are plotted in FIG. 22. A low concentrationneeded to inhibit the bacteria indicates a high potency for theantibacterial agent. The new DMAHM was more strongly antibacterial thanthe previously-synthesized QADM, requiring a lower concentration, by anorder of magnitude, to achieve the same bacteria-inhibitory effect. Thenew DMADDM was even more potent than DMAHM. The MIC and MBC of DMADDMwas three orders of magnitude lower than those of MDAHM, and four ordersof magnitude lower than QADM. The MIC and MBC of DMADDM approached thoseof CHX control.

The ADT results for un-cured primers are shown in FIG. 23. In (A), thecommercial control primer had a minimal inhibition zone, as expected.Incorporation of QADM, DMAHM and DMADDM significantly increased theinhibition zone size. In (B-D), the primer with DMADDM had significantlylarger inhibition zones than the primers with DMAHM and QADM (p<0.05).Incorporating 0.1% NAg into the primer with DMADDM further increased theinhibition zone size, which was about 10-fold those of the SBMP controlprimer (p<0.05).

The biofilm setup schematic and metabolic activity are shown in FIG. 24.In FIG. 24A, the biofilm was adherent on the cured primer covering theadhesive and the composite. In FIG. 24B, the biofilm was on the adhesivecovering the composite without a primer, for the purpose of testing theantibacterial properties of the adhesive. In FIG. 24C, the first threegroups followed the setup in FIG. 24A, groups 4 and 5 followed the setupin FIG. 24B, and the last group also followed FIG. 24A. The MTT results(mean±sd; n=6) in FIG. 24C showed that biofilms on SBMP had a highmetabolic activity. Incorporation of DMADDM and NAg into the primergreatly reduced the metabolic activity (p<0.05). Similarly,incorporating DMADDM and NAg into the adhesive also reduced themetabolic activity. In the last group, with both primer and adhesivebeing antibacterial, the lowest metabolic activity was achieved.A&P+DMADDM+NAg yielded a biofilm metabolic activity that was about20-fold lower than that of commercial bonding agent control.

FIG. 25 plots the CFU counts for: (A) Total microorganisms, (B) totalstreptococci, and (C) mutans streptococci (mean±sd; n=6). The values areshown in a log scale. Incorporation of DMADDM into the primer greatlyreduced all three CFU counts, compared to the control group (p<0.05).Combining DMADDM with NAg in the primer significantly decreased the CFUthan that using DMADDM alone (p<0.05). The same trend was observed whenDMADDM and NAg were incorporated into the adhesive.

The lactic acid production by biofilms is plotted in FIG. 26 (mean±sd;n=6). Biofilms on commercial control primer produced the most lacticacid. Adding 5% of DMADDM in either primer or adhesive greatly reducedacid production, comparing to the control (p<0.05). Using DMADDM+NAg hada significantly stronger acid-inhibiting effect than using DMADDM alone(p<0.05). Adding DMADDM+NAg in both the primer and the adhesive furtherreduced lactic acid production (p<0.05). The lactic acid production bybiofilms on P&A+DMADDM+NAg was approximately 1/20 of that on thecommercial bonding agent.

FIG. 27 plots the dentin shear bond results (mean±sd; n=10). The sixgroups had shear bond strengths that were not significantly differentfrom each other (p>0.1). This indicates that incorporation of the newquaternary ammonium monomer DMADDM and NAg into primer and adhesive toobtain antibacterial activity did not adversely affect the dentin shearbond strength.

All documents, books, manuals, papers, patents, published patentapplications, guides, abstracts and other reference materials citedherein are incorporated by reference in their entirety. While theforegoing specification teaches the principles of the present invention,with examples provided for the purpose of illustration, it will beappreciated by one skilled in the art from reading this disclosure thatvarious changes in form and detail can be made without departing fromthe true scope of the invention.

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What is claimed is:
 1. A dental primer comprising a primer and one ormore antibacterial agents, wherein one or more of the antibacterialagents is an antibacterial monomer selected from the group consisting ofdimethylamino propyl methacrylate (DMAPM), dimethylamino hexylmethacrylate (DMAHM), dimethylamino heptyl methacrylate (DMAHPM),dimethylamino octyl methacrylate (DMAOM), dimethylamino nonylmethacrylate (DMANM), dimethylamino decyl methacrylate (DMADM),dimethylamino undecyl methacrylate (DMAUDM), dimethylamino dodecylmethacrylate (DMADDM), dimethylamino tridecyl methacrylate (DMATDM),dimethylamino tetradecyl methacrylate (DMATTDM), dimethylaminopentadecyl methacrylate (DMAPDM), dimethylamino hexadecyl methacrylate(DMAHDM), dimethylamino heptadecyl methacrylate (DMAHPDM), dimethylaminooctadecyl methacrylate (DMAODM), dimethylamino nonadecyl methacrylate(DMANDM), dimethylamino icosyl methacrylate (DMAIOM), dimethylaminohenicosyl methacrylate (DMAHOM), and dimethylamino docosyl methacrylate(DMADOM).
 2. The dental primer of claim 1, wherein the primer comprisesone or more primers selected from the group consisting of bisphenol Adiglycidyl methacrylate (Bis-GMA), glycerol dimethacrylate (GDMA),2-hydroxyethyl methacrylate (HEMA), mono-2-methacryloyloxyethylphthalate (MMEP), methacrylic acid (MA), methyl methacrylate (MMA),4-acryloyloxyethyl trimellitate anhydride (4-AETA), N-phenylglycineglycidyl methacrylate (NPG-GMA), N-tolylglycine glycidyl methacrylate orN-(2-hydroxy-3-((2-methyl-1-oxo-2-propenyl)oxy)propyl)-N-tolyl glycine(NTG-GMA), pyromellitic diethylmethacrylate or2,5-dimethacryloyloxyethyloxycarbonyl-1,4-benzenedicarboxylic acid(PMDM), pyromellitic glycerol dimethacrylate or2,5-bis(1,3-dimethacryloyloxyprop-2-yloxycarbonyl)benzene-1,4-dicarboxylicacid (PMGDM), and triethylene glycol dimethacrylate (TEGDMA).
 3. Thedental primer of claim 1, wherein the primer comprises (a) 35-45%2-hydroxyethylmethacrylate (HEMA), 10-20% copolymer of acrylic anditaconic acids, 40-50% water, or the primer comprises (b) PMGDM/HEMA at3.3/1 ratio+1% BAPO+50% acetone.
 4. The dental primer of claim 1,wherein a further antibacterial agent present in the dental primer isselected from the group consisting of silver-containing nanoparticles(NAg), quaternary ammonium salts (QAS), chlorhexidine particles, TiO2particles and ZnO particles.
 5. The dental primer of claim 1, whereinthe antibacterial monomers are selected from the group consisting ofDMADDM, DMATDM, DMATTDM, DMAPDM and DMAHDM.
 6. The dental primer ofclaim 1, wherein the combined amount of antibacterial monomers presentin the dental primer ranges from about 2.5% to about 12.5% of the massof the dental primer.
 7. The dental primer of claim 4, wherein when theone or more antibacterial agents include NAg, NAg is present in thedental primer in an amount ranging from about 0.05% to about 1% of themass of the dental primer.
 8. The dental primer of claim 4, wherein whenthe one or more antibacterial agents include QAS, QAS is present in thedental primer in an amount ranging from about 3% to about 15% of themass of the dental primer.
 9. The dental primer of claim 1, furthercomprising a remineralizing agent.
 10. The dental primer of claim 9,wherein the remineralizing agent is nanoparticles of amorphous calciumphosphate (NACP) present in the dental primer in an amount ranging fromabout 5% to about 45% of the mass of the dental primer.
 11. A dentaladhesive comprising an adhesive and one or more antibacterial agents,wherein one or more of the antibacterial agents is an antibacterialmonomer selected from the group consisting of dimethylamino propylmethacrylate (DMAPM), dimethylamino hexyl methacrylate (DMAHM),dimethylamino heptyl methacrylate (DMAHPM), dimethylamino octylmethacrylate (DMAOM), dimethylamino nonyl methacrylate (DMANM),dimethylamino decyl methacrylate (DMADM), dimethylamino undecylmethacrylate (DMAUDM), dimethylamino dodecyl methacrylate (DMADDM),dimethylamino tridecyl methacrylate (DMATDM), dimethylamino tetradecylmethacrylate (DMATTDM), dimethylamino pentadecyl methacrylate (DMAPDM),dimethylamino hexadecyl methacrylate (DMAHDM), dimethylamino heptadecylmethacrylate (DMAHPDM), dimethylamino octadecyl methacrylate (DMAODM),dimethylamino nonadecyl methacrylate (DMANDM), dimethylamino icosylmethacrylate (DMAIOM), dimethylamino henicosyl methacrylate (DMAHOM),and dimethylamino docosyl methacrylate (DMADOM).
 12. The dental adhesiveof claim 11, wherein the adhesive comprises one or more adhesivesselected from the group consisting of ethoxylated bisphenol A glycoldimethacrylate (Bis-EMA), bisphenol A diglycidyl methacrylate (Bis-GMA),2-hydroxyethyl methacrylate (HEMA), triethylene glycol dimethacrylate(TEGDMA), urethane dimethacrylate (UDMA), 4-methacryloyloxyethyltrimellitate anhydride (4-META), methacrylic acid (MA), methylmethacrylate (MMA), 4-acryloyloxyethyl trimellitate anhydride (4-AETA),ethyleneglycol dimethacrylate (EGDMA), glycerol dimethacrylate (GDMA),glycerol phosphate dimethacrylate (GPDM), pyromellitic glyceroldimethacrylate or2,5-bis(1,3-dimethacryloyloxyprop-2-yloxycarbonyl)benzene-1,4-dicarboxylicacid (PMGDM).
 13. The dental adhesive of claim 11, wherein the adhesivecomprises (a) 60-70% BisGMA and 30-40% HEM, or the adhesive comprises(b) BisGMA/TEGMA at 7/3 ratio+1% BAPO.
 14. The dental adhesive of claim11, wherein a further antibacterial agent present in the dental adhesiveis selected from the group consisting of silver-containing nanoparticles(NAg), quaternary ammonium salts (QAS), chlorhexidine particles, TiO2particles and ZnO particles.
 15. The dental adhesive of claim 11,wherein when the one or more antibacterial agents includes antibacterialmonomers, the antibacterial monomers are selected from the groupconsisting of DMADDM, DMATDM, DMATTDM, DMAPDM and DMAHDM.
 16. The dentaladhesive of claim 11, wherein the combined amount of antibacterialmonomers present in the dental adhesive ranges from about 2.5% to about12.5% of the mass of the dental adhesive.
 17. The dental adhesive ofclaim 14, wherein when the one or more antibacterial agents include NAg,NAg is present in the dental adhesive in an amount ranging from about0.05% to about 1% of the mass of the dental adhesive.
 18. The dentaladhesive of claim 14, wherein when the one or more antibacterial agentsinclude QAS, QAS is present in the dental adhesive in an amount rangingfrom about 3% to about 15% of the mass of the dental adhesive.
 19. Thedental adhesive of claim 11, further comprising a remineralizing agent.20. The dental adhesive of claim 19, wherein the remineralizing agent isnanoparticles of amorphous calcium phosphate (NACP) present in thedental adhesive in an amount ranging from about 5% to about 45% of themass of the dental adhesive.